Tryptase inhibitor

ABSTRACT

PCT No. PCT/EP94/02445 Sec. 371 Date Jan. 25, 1996 Sec. 102(e) Date Jan. 25, 1996 PCT Filed Jul. 25, 1994 PCT Pub. No. WO95/03333 PCT Pub. Date Feb. 2, 1995The present invention relates to novel inhibitors of human tryptase, to their isolation from leeches, to nucleotide sequences encoding the novel inhibtor molecules or fragments thereof, to vectors containing the coding sequence thereof, to host cells transformed with such vectors, to the recombinant production of the inibitors, to pharmaceutical compositions containing the novel inhibitor molecules, and to their use in diagnosis and therapy.

The present invention relates to novel inhibitors of human tryptase totheir isolation from leeches, to nucleotide sequences encoding the novelinhibitor molecules or fragments thereof, to vectors containing thecoding sequence thereof, to host cells transformed with such vectors, tothe recombinant production of the inhibitors, to pharmaceuticalcompositions containing the novel inhibitor molecules, and to their usein diagnosis and therapy.

Tryptase is a tetrameric member of the family of trypsin-like serineproteinases. Tryptase is expressed virtually exclusively by mast cells[Castells Irani, 1987] and stored in large amounts in their secretorygranules, constituting ˜23% of the total cellular protein [SchwartzLewis Austen, 1981]. Following activation of mast cells, tryptase israpidly released into the extracellular space together with otherpreformed mediators (e.g. histamine, chymase, and proteoglycans)[Schwartz Lewis Seldin, 1981; Caughey Lazarus, 1988]. Elevated levelshave been found

in the plasma of patients with mastocytosis, after systemic anaphylaxis[Schwartz Metcalfe, 1987; Schwartz Yunginger, 1989], and during thesystemic response after aspirin challenge of patients withaspirin-sensitive asthma [Bosso Schwartz, 1991],

in bronchoalveolar lavage fluid of patients with asthma [Broide Gleich,1991; Bousquet Chanez, 1991; Wenzel Fowler, 1988], interstitial lungdiseases [Walls Bennett, 1991], and after antigen challenge of allergicpatients [Castells, 1988; Butrus, 1990],

in the skin blister fluid after cutaneous antigen challenge in patientswith atopic and allergic skin disease [Shalit Schwartz, 1990; AtkinsSchwartz, 1990],

in nasal lavage fluid after local antigen challenge of patients withseasonal allergic rhinitis [Juliusson Holmberg, 1991],

in the crevicular fluid of patients with gingivitis and periodontitis[Cox Eley, 1989, J Period Res; Eley Cox, 1992, J Dent], and

in the lesional skin of patients with psoriasis [Harvima Naukkarinen,1989].

In vitro studies have provided considerable evidence that tryptase isdirectly involved in the pathogenesis of mast cell related disorders.For example, tryptase has been suggested as a pathogenetic mediator ofasthma as it increases the contractility of airway smooth muscle[Sekizawa, 1989] and inactivates vasoactive intestinal peptide, therebydestroying its potent bronchodilatatory action [Tam Caughey, 1990; TamFranconi, 1990; Franconi, 1989]. In addition, tryptase has been shown tobe a potent mitogen for fibroblasts, suggesting its involvement in thepulmonary fibrosis in asthma and interstitial lung diseases [RuossHartmann, 1991; Hartmann Ruoss, 1992]. Tryptase has also been implicatedin the pathogenesis of arthritis and periodontal disease, as itactivates prostromelysin (=MMP-3) which in turn activates collagenase,thereby initiating the destruction of cartilage and periodontalconnective tissue, respectively [Gruber Marchese, 1989; Gruber Schwartz,1990; Cox Eley, 1989, J Period Res; Eley Cox, 1992, J Dent]. Tryptasemay also promote blood clotting disorders by inactivating theprocoagulant function of high molecular weight kininogen [Maier Spragg,1983] and by cleaving fibrinogen [Schwartz Bradford Littman, 1985].

Human tryptase is virtually unique among the serine proteinases as it isfully catalytically active in plasma and in the extracellular space[Schwartz Bradford, 1986; Goldstein Leong, 1992]. Tryptase is notinhibited by the naturally occurring antiproteinases regulating theactivity of other trypsin-like serine proteinases such asmucus-proteinase inhibitor (=antileukoprotease or HUSI-I), antithrombinIII, alpha₁ -proteinase inhibitor, alpha₂ -macroglobulin, or C₁-esterase inhibitor [Alter Kramps, 1990; Smith Hougland, 1984; SchwartzBradford, 1986; Harvima Schechter, 1988; Cromlish Seidah, 19873].Furthermore, although tryptase has been known for over 10 years,inhibitors derived from non-human species or produced by peptidesynthesis or recombinant technologies have not yet been described. Thus,tryptase is not affected by hirudin [Alter Kramps, 1990], aprotinin,ovomucoid inhibitor, soybean and lima bean trypsin inhibitor[Butterfield Weiler, 1990; Cromlish Seidah, 1987; Harvima Schechter,1988], ecotin [Chung Ives, 1983], and the recombinant Kunitz-domain ofthe Alzheimer beta-amyloid precursor-protein [Sinha Dovey, 1990].

Although tryptase is inhibited by the general inhibitors of trypsin-likeproteinases such as diisopropyl fluorophosphate, phenylmethylsulfonylfluoride and tosyl-L-lysine chloromethyl ketone [Smith Hougland, 1984;Harvima Schechter, 1988], these compounds are unsuitable for in vivo andeven for most in vitro applications due to their high toxicity and/orlow stability. Furthermore, the only other inhibitors known to affecttryptase, the peptide-arginine aldehydes leupeptin and antipain[Cromlish Seidah, 1987], and certain benzamidin derivatives[Sturzebecher Prasa, 1992; Caughey, 1993] are of limited usefulness asthey are relatively unspecific, and/or inhibit tryptase only withmoderate affinities (K_(i) values for the complexes in the micromolarrange).

The problem of the present invention is therefore to provide a potentand efficient inhibitor of the human proteinase tryptase.

As illustrated in further detail below the present problem can be solvedby providing an inhibitor polypeptide obtainable from medical leechHirudo medicinalis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Isolation of the leech-derived tryptase inhibitor bycation-exchange chromatography using SP-Sephadex. Dialysed leech extractwas applied and the column was washed until the absorption of theeffluent returned to baseline. Desorption was achieved with 20 mM NaP,500 mM NaCl (pH 8.0). Fractions containing inhibitory active material(marked with a bar) were pooled.

FIG. 2: Affinity-chromatography of the leech-derived tryptase inhibitorusing anhydrotrypsin-Sepharose. The pooled eluate from the SP-Sephadexchromatography was applied, the column washed extensively and elutedwith 100 mM KCl/HCl (pH 2.1). Fractions containing inhibitory activematerial (marked with a bar) were collected and neutralised immediatelyby addition of 1 M Tris.

FIG. 3: Cation-exchange chromatography of the leech-derived tryptaseinhibitor using a Mono S FPLC column. After dialysis against 20 mM NaP(pH 8.0) the pooled eluate from the anhydrotrypsinaffinity-chromatography was applied, the column washed, and eluted usinga linear gradient from 60 to 240 mM NaCl. The fractions containinginhibitory active material (marked with a bar) were pooled.

FIG. 4: SDS-PAGE of the isolated leech-derived tryptase inhibitor underreducing conditions. Lane 1=dialysed leech extract; lane 2=eluate fromthe SP-Sephadex column; lane 3=eluate from the anhydrotrypsinaffinity-chromatography; lane 4=eluate from Mono S cation exchangechromatography. The molecular weight markers (lane 5) are from top tobottom: ovalbumin (43 kD), carbonic anhydrase (29 kD), β-lactoglobulin(18.4 kD), lysozyme (14.3 kD), bovine trypsin inhibitor (6200 D), andinsulin β-chain (3400 D).

FIG. 5: Reversed phase HPLC of the isolated leech-derived tryptaseinhibitor. The elution time and the absorption of the effluent at 206 nmare given on the abscissa and the ordinate, respectively. The two peaksdemonstrate the presence of at least two forms.

FIG. 6: Tryptic fragmentation of the two species of the leech-derivedtryptase inhibitor separated by reversed phase HPLC (see FIG. 5). Lowertracing: HPLC-tracing of the tryptic digest of the peak eluting at 25min in FIG. 5; Upper tracing: HPLC-tracing of the tryptic digest of thepeak eluting at 29 min. The elution profiles differ only in the peaksrepresenting the C-termial peptides (marked by arrows).

FIGS. 7A and 7B: Mass spectroscopy of the two species of theleech-derived tryptase inhibitor separated by HPLC (see FIG. 5). a) Themass spectrum of the HPLC peak eluting at 25 min demonstrates thepresence of 2 forms with a mass of 4340 (form A; left peaks) and 4396(form B; right peaks), respectively. b) The mass spectrum of the HPLCpeak eluting at 29 min shows a third form (form C) with a mass of 4738.

FIG. 8: Sequence determination of the leech-derived tryptase-inhibitor.The bars represent the overlapping fragments used in deducing the aminoacid sequence. The solid bars denote the sequence obtained from the HPLCpeak eluting at 25 min (see FIG. 5), and the hatched bar the additionalsequence obtained from the HPLC peak eluting at 29 min.N-terminal=sequence obtained from the native inhibitor; Red/T=sequenceobtained after reduction and tryptic fragmentation; Ox/T/ChT=sequenceobtained after oxidation and tryptic/chymotryptic fragmentation.

FIG. 9: Inhibition of human tryptase by the leech-derived tryptaseinhibitor. Tryptase (0.59 nM) was preincubated with increasingconcentrations of the leech-derived tryptase inhibitor (0-40 nM) at 37°C. for 25 min, and the reaction was initiated by the addition ofsubstrate tos-Gly-Pro-Arg-pNa. The resulting steady state velocitieswere measured over 3.5 min. The values given are the quotient of thevelocity in the presence of the inhibitor divided by the velocity in theabsence of the inhibitor.

FIG. 10: Effect of the leech-derived tryptase inhibitor on thetryptase-induced cleavage of vasoactive intestinal peptide (VIP). VIPwas incubated with tryptase in the presence of increasing concentrationsof the leech derived tryptase inhibitor. Thereafter, the amount of VIPcleaved was quantified by reversed phase HPLC. The values given are thequotient of the velocity in the presence of the inhibitor divided by thevelocity in the absence of the inhibitor.

FIGS. 11A-11C: Design, DNA and amino acid sequence of synthetic rLDTIform-C gene. (a) Design of the synthetic rLDTI form-C master gene. Theintroduced restriction sites are shown. (b) Nucleotide and correspondingamino acid sequence of the rLDTI form-C master gene. Brackets andnumbers indicate the synthetic oligonucleotides used to assemble thegene. (c) Modification of rLDTI form-C master gene by cassettemutagenesis

FIGS. 12A and 12B: (a) Plasmid map of pRM 5.1.5. (b) Expression vectorpRM 9.1.4. A synthetic gene for rLDTI-form C was ligated into thepurified yeast secretion vector pVT102U/α, cleaved with XbaI andHindIII. Arrows indicate the direction of transcription; ADH-p, the ADH1gene promotor; mat, the α-mating factor leader gene; ADH-t, the 3'region of the ADH1 gene including a transcription terminator signal;Ura-3, the Ura gene; amp-R, the ampicillin-resistence gene; the E.coliori, yeast ori (2μ-ori) and the intergenic region of phage f1 (f1-ori).

FIG. 13: SDS/PAGE analysis of fermentation supernatant and purifiedrLDTI form-C. lane 1=low-molecular mass-markers; lane 2=fermentationsupernatant of yeast strain HOO5 after 96 hours of cultivation (80 μl);lane 3=purified rLDTI-form C (2 μg).

FIG. 14: HPLC analysis of purified rLDTI form-C. Reverse phase HPLC on aRP 18 column was performed with 7,4 nmol (35 μg) purified inhibitor. Alinear gradient of 0-60% (by vol.) acetonitrile formed from 0,1% (byvol.) trifluoroacetic acid in acetonitrile and 0,1% (by vol.)trifluoroacetic acid was used. The flow rate was adjusted to 1.0 ml/minand the absorbance in the effluent was monitored at 206 nm.

FIG. 15: Plasmid map of expression vector pRM 3.1.10

FIG. 16: Plasmid map of expression vector pRM 4.1.4

FIG. 17: Plasmid map of expression vector pRM 11.1.4.

According to a first embodiment the present invention relates topurified inhibitor molecules of human tryptase. The novel inhibitors arepolypeptides obtainable from extracts of leeches, as for example themedical leech Hirudo medicinalis. The invention also relates to thefunctional equivalents of the inhibitor molecules showing tryptaseinhibitor activity, and to the pharmaceutically acceptable salts of theinhibitors.

The inhibitor molecules of the present invention are characterized bytheir ability to inhibit human tryptase with a K_(i) value in the rangeof approximately 0.1 to 10 nM, leaving the proteases involved in thehuman blood coagulation cascade substantially unaffected.

Preferably tryptase inhibitors are provided which are characterized bythe following amino acid sequence (position 1: N-terminal amino acidLys):

    Lys-Lys-Val-Cys-Ala-Cys-Pro-Lys-Ile-Leu 10                                    Lys-Pro-Val-Cys-Gly-Ser-Asp-Gly-Arg-Thr 20                                    Tyr-Ala-Asn-Ser-Cys-Ile-Ala-Arg-Cys-Asn 30                                    Gly-Val-Ser-Ile-Lys-Ser-Glu-Gly-Ser-Cys 40                                    Pro-Thr-X                               42                                

wherein X=H (SEQ ID No:1), Gly (SEQ ID NO:2) or Gly-Ile-Leu-Asn (SEQ IDNO:3).

The present invention also encompasses genetic variants, alleles orfunctional equivalents of the above-mentioned sequence, of which one ormore of the amino acids are substituted (conservative ornon-conservative) or deleted, or to which one or more amino acids areadded without substantially affecting the tryptase inhibitor activity.Conservative substitutions encompass for example substitutions withinthe following groups of amino acids (one letter code): G,A; V,I,L; D,E;N,Q; K,R; and S,Y,N. Preferebly the amino acid addition or deletion isperformed at the N- and/or C-terminal end of the above-mentionedsequence. The functional equivalents with altered and/or improvedspecificity and/or inhibitory efficiency can easily be prepared by aperson of ordinary skill applying usual methods of peptide synthesis, orapplying methods well known in the field of molecular biology, as forexample site directed mutagenesis or undirected mutagenesis (e.g. usinga phage display system).

Functional equivalents of the inhibitor of the present invention are forexample those comprising the amino acid sequence (SEQ ID NO:21)

                   R.sup.1 -Cys-Pro-Lys-Ile-Leu                                   Lys-Pro-Val-Z-Gly-Ser-Asp-Gly-Arg-Thr                                         Tyr-Ala-Asn-Ser-Cys-Ile-Ala-R.sup.2                                       

wherein

the N-terminal residue R¹ represents Ala- or Cys-Ala-;

the C-terminal residue R² represents -Arg or -Arg-Cys; and

Z defines any, preferably any naturally occurring, amino acid.

Moreover, based on the teaching of the present invention a person ofordinary skill will be enabled to prepare fragments of the natural formsof the inhibitor still showing the desired tryptase inhibiting activity.

The naturally occurring forms of the claimed inhibitor molecules mayeither be isolated from leech, preferably the medical leech Hirudomedicinalis, or may be prepared by peptide synthesis or recombinant DNAtechnology.

According to a further aspect of the present invention a synthetic genecoding for form C (SEQ ID NO:3) of the leech derived tryptase inhibitor(LDTI-C) was designed, cloned and expressed in Escherichia coli andSaccharomyces cerevisiae. The coding fragment was assembled via 6oligonucleotides, it contains linker sequences, stop codons and selectedrecognition sites for further modifications, for example by cassettemutagenesis. Strong expression of the recombinant form C inhibitor(rLDTI-C) was found using Saccharomyces cerevisiae secretion vectorpVT102U/alpha and strain S-78. The secreted material was isolated bycentrifugation and cross-flow filtration, and further purified by cationexchange chromatography, it is inhibitorily active and about 85% pure.Amino acid sequencing showed that rLDTI-C is predominantly correctprocessed at the junction between the alpha mating factor leader peptideand the first amino acids of LDTI-C; only minor amounts of truncatedforms were detected. The far UV-CD spectrum of the recombinant moleculeis typical for a folded protein containing secondary structuralelements. The molecular mass of HPLC purified material is 4738±4 Da asdetermined by electrospray ionization mass spectrometry. The rLDTI-Cdisplays equilibrium dissociation constants with bovine trypsin andhuman tryptase which are nearly identical to those of the natural one.The expected expression products encoded within the expression vectorwere also identified in vitro, using a S30 transcription translationsystem.

The proteins presented in this invention are the first compounds knownto be efficient inhibitors of tryptase. Thus, the leech-derived tryptaseinhibitors reduce the catalytic activity of tryptase, the K_(i) value ofthe enzyme-inhibitor complex being in the nanomolar range. Moreover, theinhibitors affect not only the tryptase-induced cleavage of thepeptide-nitroanilid substrate used as a tool to determine the activityof the proteinase in vitro. They also affect the cleavage of vasoactiveintestinal peptide (VIP) and of kininogen, representatives of thepeptides and proteins thought to be biologically relevant substrates oftryptase. In addition, the inhibitors efficiently diminish thetryptase-induced growth of human keratinocytes--an example of the directcellular effects of tryptase--without causing apparent cytotoxic orother side effects.

Besides having a high affinity for tryptase, the leech-derived tryptaseinhibitors are highly specific. Thus, with the exception of thepancreatic proteinases trypsin and chymotrypsin, other serineproteinases are not or only marginally inhibited, the K_(i) values forthe enzyme-inhibitor complexes being at least 200 times higher than forthe complex with tryptase. Their specificity is illustrated by thelacking effect on the blood coagulation ex vivo, verifying that theproteinases involved in the coagulation cascade are not affected.

Thus, the leech-derived tryptase inhibitors of the present inventionwill allow for the first time the inhibition of tryptase with highaffinity and specificity. Consequently, the inhibitors provide theprospect to effectively block pathophysiologic events involving thecleavage of proteins and peptides and/or the activation of cells bytryptase.

Therefore, it is an object of the current invention to apply theinhibitors as probes in the diagnosis well as drugs in the therapy oftryptase- and mast cell related diseases.

According to a further preferred embodiment of the present inventionnucleotide sequences, as for example DNA and RNA sequences, are providedwhich encode a polypeptide with tryptase inhibitor activity or fragmentsthereof. Preferably polynucleotide molecules comprising the followinggeneral nucleotide sequence (SEQ ID NO:4) are provided:

                                      5'                                                                          1 AARAARGTNTGYGCNTGYCCNAARATHYTNAARCCNGTNT                                  GYGGNWSNGA                                                                     51 YGGNMGNACNTAYGCNAAYWSNTGYATHGCNMGNTGYAAY                                  GGNGTNWSNA                                                                    101 THAARWSNGARGGNWSNTGYCCNACNX                                                                            3'             

wherein R denotes A or G; M denotes A or C; W denotes A or T; S denotesC or G; Y denotes C or T; H denotes A, C, or T; N denotes anynucleotide; and X denotes --OH (SEQ ID NO:4), GGN (SEQ ID NO:5) or GGNATH YTN AAY (SEQ ID NO:6). The invention also relates to thecomplementary strand thereof; and the DNA sequences which hybridize,preferably under stringent conditions, to the afore-mentioned DNAsequence.

Preferably the polynucleotides of the present invention comprise anucleotide sequence substantially corresponding to nucleotide residues 1to 149, or more preferably 7 to 144 of SEQ ID NO:7; or fragments thereofcomprising at least 15 to 21 consecutive nucleotides of SEQ ID NO:7.Within the scope of the invention are also complementary polynucleotidescomprising a nucleotide sequence substantially corresponding tonucleotide residues 1 to 149, or preferably 10 to 147 of SEQ ID NO:8; orfragments thereof comprising at least 15 to 21 consecutive nucleotidesof SEQ ID NO:8. Preferably, these fragments are tryptase inhibitorspecific or functional derivatives of these nucleotide sequences.

According to a further embodiment the present invention refers to anoligonucleotide which hybridizes, preferably under stringent conditions,to a nucleotide sequence encoding a polypeptide with tryptase inhibitoractivity. Preferably, this oligonucleotide comprises a nucleotidesequence which is substantially complementary to the nucleotide sequencefrom residue 22 to residue 87 of SEQ ID NO:7.

Another embodiment of the invention refers to polynucleotides encoding apolypeptide with tryptase inhibitor activity which polynucleotides beingobtainable by hybridizing, preferably under stringent conditions, withan oligonucleotide as specified above; as well as to polypeptidesencoded by said polynucleotides. Suitable stringent conditions can bedetermined easily by one skilled in the art.

Also within the scope of the present invention are the polynucleotidesequence of SEQ ID NO:9, and functional equivalents thereof.

The present invention also encompasses vector molecules for thetransformation of eucaryotic or procaryotic hosts, comprising a DNAmolecule as defined above. For example, the vector may be a virus or aplasmid containing the inhibitor encoding DNA sequence in functionalrelation with suitable transcriptional and translational regulatorysequences well known in the art. The coding sequence may also be linkedto suitable autonomously replicating sequences (ARS). Suitable hostcells may be transformed with a vector containing the tryptase inhibitorcoding sequence, and the inhibitor produced by the host cells may beexpressed and isolated in a suitable way from the the cell culture.

A further embodiment of the invention is a polypeptide expressioncassette comprising a promoter operably linked to a DNA sequence codingfor the polypeptide and to a DNA sequence containing transcriptiontermination signals. In hosts capable of secreting expressedpolypeptides, the expression cassette preferably comprises a promoteroperably linked to a first DNA sequence encoding a signal peptide linkedin the proper reading frame to a second DNA sequence coding for theinventive polypeptide, and a DNA sequence containing transcriptiontermination signals.

In a preferred embodiment, the promoter, the signal sequence and theterminator are recognized by the yeast expression system.

Promoters suitable for expression in a certain host are well known.Examples are the promoter of the TRP1 gene, the ADHI or ADHII gene, acidphosphatase (PHO5) gene, CUP1 gene, iso-cytochrome c gene, or a promoterof the genes coding for glycolytic enzymes, such as TDH3,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a shortened version ofGAPDH (GAPFL), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, invertase and glucokinase genes, or a promoterof the yeast mating pheromone genes coding for the a- or α-factor, canbe used. Preferred vectors of the present invention contain, e.g.,promoters with transcriptional control that can be turned on or off byvariation of the growth conditions, e.g. the promoter of the PHO5 or theCUP1 gene. For example, the PHO5 promoter can be repressed orderepressed at will, solely by increasing or decreasing theconcentration of inorganic phosphate in the medium and the CUP1 promotercan be turned on by the addition of Cu²⁺ -ions to the medium, e.g., inthe form of a copper salt. Especially preferred are the GAPDH and theyeast CUP1 promoter.

The DNA sequence encoding a signal peptide ("signal sequence"), e.g. ayeast signal peptide, is preferably derived from a gene, e.g. a yeastgene, coding for a polypeptide which is ordinarily secreted. Yeastsignal sequences are, for example, the signal and prepro sequences ofthe yeast invertase (SUC2), α-factor, pheromone peptidase (KEX1),"killer toxin" and repressible acid phosphatase (PHO5) genes and theglucoamylase signal sequence from Aspergillus awamori. Additionalsequences, such as pro- or spacer-sequences which may carry specificprocessing signals can also be included in the constructions tofacilitate accurate processing of precursor molecules. For example, theprocessing signals contain a Lys-Arg residue, which is recognized by ayeast endopeptidase located in the Golgi membranes. The preferred signalsequences according to the present invention are those of the yeast PHO5gene, the α-factor and of the yeast invertase gene (SUC2).

A DNA sequence containing transcription termination signals, e.g. yeasttranscription termination signals, is preferably the 3' flankingsequence of a gene, e.g. a yeast gene, which contains proper signals fortranscription termination and polyadenylation. The preferred flankingsequence is that of the yeast PHO5 and the α-factor gene.

The DNA coding for the polypeptide according to the invention may beisolated from a gene bank of the natural host (the medical leech Hirudomedicinalis) by methods known in the art or synthesized by PCR using,e.g., the preferred codon usage of the host.

The promoter, the DNA sequence coding for the signal peptide, the DNAsequence coding for the polypeptide and the DNA sequence containingtranscription termination signals are operably linked to each other,i.e. they are juxtaposed in such a manner that their normal functionsare maintained. The array is such that the promoter effects properexpression of the signal sequence-polypeptide gene complex, thetranscription termination signals effect proper termination oftranscription and polyadenylation. The signal sequence is linked in theproper reading frame to the polypeptide gene in such a manner that thelast codon of the signal sequence is directly linked to the first codonof the gene for the polypeptide. The yeast promoter is preferably joinedto the signal sequence between the major mRNA start and the ATGnaturally linked to the promoter gene. The signal sequence has its ownATG for translation initiation. The junction of these sequences may, forexample, be effected by means of synthetic oligo-deoxynucleotide linkerscarrying the recognition sequence of an endonuclease. Examples forrelated expression cassettes are described e.g. in EP-A-341215.

Preferred expression cassettes comprise the CUP1 or the GAPDH promoter,the α-factor or the yeast invertase leader sequence, the tryptaseinhibitor gene and the α-factor terminator.

Especially preferred expression cassette comprise a recombinant DNAmolecule as described in Example 9 or a functional fragment orderivative thereof.

A further embodiment of the invention concerns a recombinant plasmidcomprising a polypeptide expression cassette as described above.

Apart from the polypeptide expression cassette the expression plasmidsaccording to the invention can comprise a DNA segment originating fromtwo-micron DNA containing the origin of replication or, if a two-micronDNA free strain of yeast is used, total two-micron DNA. The latter typeof plasmids is preferred. For example, the plasmids according to theinvention may contain the complete two-micron DNA in an uninterruptedform, i.e. two-micron DNA is cleaved once with a restrictionendonuclease, the linearized DNA is linked with the other components ofthe vector prior to recircularization. The restriction site is chosensuch that normal function of the REP1, REP2 and FLP genes and of theORI, STB, IR1 and IR2 sites of two-micron DNA as well as small "FLPrecognition target" (FRT) sites, located near the center of eachinverted repeat (IR) at which the FLP recombinase acts, is maintained.Optionally, the restriction site is chosen such that the D gene oftwo-micron DNA is kept intact, too. Suitable restriction sites are, forexample, the unique PstI site located within the D gene and the uniqueHpaI and SnaBI sites located outside of all of said genes and sites.However, it is likewise possible to insert the expression cassette andfurther components (cf. below) at different (such as two) restrictionsites, especially those mentioned above, within two-micron DNA.

Preferably, the expression plasmids according to the invention includeone or more, especially one or two, selective genetic markers, e.g. amarker for yeast and a marker and (except for symmetric two-micron likehybrid vectors) an origin of replication for a bacterial host,especially Escherichia coli.

As to the selective gene markers, any marker gene can be used whichfacilitates the selection for transformants due to the phenotypicexpression of the marker gene. Suitable markers are, for example, thoseexpressing antibiotic resistance or, in the case of auxotrophic yeastmutants, genes which complement host lesions. Corresponding genesconfer, for example, resistance to the antibiotics G418, hygromycin orbleomycin or provide for prototrophy in an auxotrophic yeast mutant, forexample the URA3, LEU2, LYS2, HIS3 or TRP1 gene.

As the amplification of the expression plasmids is conveniently done ina prokaryote, such as E. coli, a prokaryote, e.g. E. coli, geneticmarker and a prokaryote, e.g. E. coli, replication origin are includedadvantageously. These can be obtained from corresponding prokaryoticplasmids, for example E. coli plasmids, such as pBR322 or a pUC plasmid,for example pUC18 or pUC19, which contain both prokaryotic, e.g. E.coli, replication origin and genetic marker conferring resistance toantibiotics, such as ampicillin.

A suitable vector for transforming yeast cells is plasmid pRM 9.1.4 asdeposited with the bSM and having the accession number DSM 9271.

Further preferred vector molecules are

pRM11.1.4 as deposited with the DSM and having the accession number DSM9272;

pRM5.1.5 as deposited with the DSM and having the accession number DSM9270;

pRM4.1.4 as deposited with the DSM and having the accession number DSM9269; and

pRM3.1.10 as deposited with the DSM and having the accession number DSM9268.

The vector may also be selected from pHE175, pHE175R, pHE177 and pHE177Ras disclosed in the experimental part below.

According to a further embodiment of the present invention a method ofpreparing a tryptase inhibitor is provided, comprising the steps of

a) obtaining an extract of a leech, preferably of the medicinal leechHirudo medicinalis, and

b) purifying the extract by dialysis and column chromatography.

Preferably the crude extract is dialyzed against a buffer of low ionicstrength. Subsequently the dialyzed extract is purified by cationexchange chromatographie, bio-specific chromatography, as for exampleanhydrotrypsin-sepharose affinity chromatography, and a further cationexchange chromatography step. Further experimental details areillustrated in the experimental part below. Modifications of the claimedprocess can easily be designed by a person of ordinary skill which arestill encompassed by the scope of the present invention.

The invention also refers to methods of preparing recombinant tryptaseinhibitor and to recombinant tryptase inhibitors obtainable by thesemethods. According to one preferred embodiment this method comprises

a) transforming a prokaryotic or eukaryotic host with a vector asdefined above;

b) inducing the expression of the tryptase inhibitor encoding sequence;

c) recovering the expression product; and optionally

d) removing from the obtained product peptide fragments not required fortryptase inhibitor activity and/or optionally renaturing the product.

The coding sequence may also be expressed applying a suitabletranscription translation system, as for example the S30 transcriptiontranslation system.

According to another embodiment of the present invention prokaryotic andeukaryotic hosts transformed with a vector encoding a tryptaseinhibitor, and variants and mutants thereof are provided.

Suitable hosts are of prokaryotic or eukaryotic origin. Examples arebacterial, fungal, plant or insect cells. preferred hosts are bacterialand fungal cells such as E. coli or fungi like Saccharomyces cerevisiae,Aspergillus niger, Aspergillus nidulans or Neurospora crassa.

Preferred yeast strains are those mentioned above, e.g. strains of S.cerevisiae which have been cured of the endogenous two-micron plasmid("cir.sup.° strains") and especially strains which are singly ormultiply deficient in yeast proteases; and/or, in the case the CUP1promoter is used, yeast strains containing 1-3 additional copies of thechromosomal CUP1 gene.

A wide variety of proteinases, like those mentioned, have beencharacterized in the yeast Saccharomyces cerevisiae [Achstetter et al.(1985)]. Mutants lacking activity of most of these proteases have beenisolated and studied biochemically. The consequences of the absence ofcertain proteases were elucidated and some properties proved to beuseful for the production of heterogeneous proteins. The proteases whichare lacking in the yeast strains according to the invention do notperform indispensible functions in the cell metabolism; thereforemutations which completely destroy the activity of these proteins arenot lethal. For example, the yeast strain lack one or more proteasesselected from the group of carboxypeptidases yscα-, yscB, yscA, yscY andyscS. Methods for the production of such yeast strains are described,for example, in EP-A-40170 and EP-A-341215.

The transformation of host with the hybrid plasmids according to theinvention may be accomplished according to methods known in the art.

A preferred embodiment refers to a eukaryotic host derived from S.cerevisiae S-78 as deposited with the DSM and having the accessionnumber DSM 9273 and to variants and mutants thereof capable of producinga tryptase inhibiting molecule.

According to another preferred embodiment of the present inventionpharmaceutical compositions are provided, comprising a tryptaseinhibiting amount of a polypeptide as defined above, prepared from leechextracts or obtained for example by expression of a recombinant tryptaseinhibitor encoding gene, optionally in combination with apharmaceutically acceptable carrier or diluent.

These compositions can be used in particular in the case of theindications mentioned herein, if they are administered, e.g.parenterally (such as intravenously, intracutaneously, intramuscularlyor subcutaneously), orally, by inhalation or topically. The dosagedepends essentially on the specific method of administration and on thepurpose of the treatment or prophylaxis. The size of the individualdoses and the administration programme can best be determined based onan individual assessment of the relevant case. The methods required todetermine the relevant factors are familiar to the expert. Normally, inthe case of injection, the therapeutically active quantity of thecompounds according to the invention is in the dosage range ofapproximately 0.005 to approximately 1 mg/kg of body weight. The rangefrom approximately 0.01 to approximately 0.05 mg/kg of body weight ispreferred.

Administration is by intravenous, intramuscular or subcutaneousinjection. consequently, depending on the method of application,pharmaceutical preparations for parenteral administration containapproximately 0.5 to approximately 10 mg of the compound according tothe invention per individual dose. In addition to the active ingredient,these pharmaceutical compositions usually also contain a buffer, e.g. aphosphate buffer, intended to keep the pH value between approximately3.5 and 7, and, furthermore, sodium chloride, mannitol or sorbitol inorder to adjust isotonicity. They can be in freeze-dried or dissolvedform, wherein the solutions can advantageously contain an antibacterialpreserving agent, e.g. 0.2 to 0.3% of 4-hydroxybenzoic acid methyl esteror ethyl ester.

A preparation for topical application can be in the form of an aqueoussolution, lotion or jelly, an oily solution or suspension or a fatty or,particularly, emulsion ointment. A preparation in the form of an aqueoussolution is obtained, e.g. by dissolving the substance according to theinvention or a therapeutically useful salt thereof in an aqueous buffersolution of pH 4 to 6.5 and, if desired, adding one or more furthersubstance thereto. The concentration of the active ingredient isapproximately 0.08 to approximately 1.5 mg, preferably 0.25 to 1.0 mg,in approximately 10 ml of a solution or 10 g of a jelly.

An oily form of application for topical administration is obtained, e.g.by suspending the substance according to the invention or atherapeutically useful salt thereof in an oil, optionally with theaddition of swelling agents, such as aluminium stearate, and/orsurface-active agents (surfactants) the HLB value(hydrophilic-lipophilic balance) of which is less than 10, such as fattyacid monoesters of polhydric alcohols, e.g. glycerol monostearate,sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. Agreasy ointment is obtained, e.g. by suspending the substance accordingto the invention or the salts in a spreadable greasy base, optionallywith the addition of a surfactant having an HLB value of less than 10.An emulsion ointment is obtained by trituration of an aqueous solutionof the substance according to the invention or the salts in a softspreadable greasy base with the addition of a surfactant, the HLB valueof which is less than 10. All of these forms of topical application canalso contain a preserving agent. The concentration of the activeingredient is approximately 0.08 to approximately 1.5 mg, preferably0.25 to 1.9 mg in approximately 10 g of the matrix.

This invention also relates to the bioanalytical use of the compoundsaccording to the invention and the salts thereof for the analyticaldetermination of trypase and the preparations serving to this end,containing the substances according to the invention, e.g. solidmixtures and above all solutions, in particular aqueous solutions. Inaddition to a specific quantity or concentration of the substancesaccording to the invention (also in the form of a salt), these can alsocontain inert adjuvants, e.g. those mentioned above with reference tothe injection preparations, which have, e.g. a stabilising and/orpreserving function.

According to a further embodiment the present invention is concernedwith the use of a tryptase inhibitor as defined above in diagnosingfunctional tryptase and mast cell related disorders. Especiallypreferred is the use for preparing pharmaceutical compositions for thetreatment of asthma, intestinal lung disease, arthritis, periodontaldisease, allergic disorders, blood clotting disorders, skin disordersand psoriasis.

EXPERIMENTAL PART

1. MATERIALS

a) Leech Extracts: Extracts from the medical leech Hirudo medicinaliswere a gift from Plantorgan, Germany. The leech extracts may also beprepared on the basis of the disclosure of EP-A-0 207 956 and thereferences cited therein.

b) Enzymes and Substrates: Proteases were obtained as follows: bovinetrypsin, porcine pancreatic kallikrein, and porcine pancreatic elastase(Sigma; Deisenhofen, Germany); human factor Xa (Boehringer Mannheim;Mannheim, Germany); human neutrophil elastase, human thrombin, humanurokinase, and bovine chymotrypsin (Medor; Herrsching, Germany); humanplasmin, and human plasma kallikrein (Kabi; Essen, Germany); humancathepsin G (Calbiochem; Bad Soden, Germany).

Tryptase was purified from human lung tissue to apparent homogeneityusing a modification of described methods [Smith Hougland, 1984;Schwartz Lewis Austen, 1981; Harvima Schechter, 1988].

The following substrates were purchased: Bz-Ile-Glu-Gly-Arg-pNA(Novabiochem; Bad Soden, Germany); Suc-Ala-Ala-Ala-pNA (Bachem;Heidelberg, Germany); D-Pro-Phe-Arg-pNA, and D-Val-Leu-Arg-pNA (Kabi;Essen, Germany); Suc-Val-Pro-Phe-pNA, and Pyr-Gly-Arg-pNA (Medor;Herrsching, Germany); MeO-Suc-Ala-Ala-Pro-Val-pNA (Sigma; Munich,Germany). Tos-Gly-Pro-Arg-pNA was obtained from Boehringer Mannheim,Medor, and Sigma. (Tos=tosyl; Suc=succinyl; pNA=p-nitroanilide).

Vasoactive intestinal peptide (VIP) was purchased from Calbiochem (BadSoden, Germany), and bovine lung heparin from Sigma. Bdellin B was agift from E. Fink (Klinische Chemie und Klinische Biochemie,Chirurgische Klinik, LMU; Munich, Germany).

c) Column materials: SP-Sephadex®, cyanogen bromide-activated Sepharose®4B and Mono S® HR 5/5 were obtained from Pharmacia (Freiburg, Germany).

Anhydrotrypsin was prepared from trypsin, affinity-purified by amodification of the methods described by Ako [Ako Foster Ryan, 1972],and immobilised onto cyanogen bromide-activated Sepharose 4B accordingto the guidelines of Pharmacia.

d) Cell culture: Media, foetal calf serum, and antibiotics were obtainedfrom Biochrom (Berlin, Germany). The human keratinocyte cell line HaCaT[Boukamp Petrussevska, 1988] was obtained from N. Fusenig, German CancerResearch Center (DKFZ; Heidelberg, Germany). [Methyl-³ H]thymidine waspurchased from Amersham Buchler (Braunschweig, Germany).

2. METHODS

2.1. Purification of the Leech-derived Tryptase Inhibitor

a) Chromatography on SP-Sephadex®: Leech extract (˜3.5 g) was dissolvedin deionised water (77 ml) and dialysed against 20 mM NaP (pH 8.0) overnight at 4° C. The dialysed material was applied onto a SP-Sephadex®column (1.6×20 cm) equilibrated with the same buffer. The column waswashed at a flow rate of 1 ml/min until the optical density (280 nm) ofthe effluent reached baseline, and eluted with 20 mM NaP, 500 mM NaCl(pH 8.0). Fractions containing inhibitory active material were collectedand pooled.

b) Affinity-chromatography on anhydrotrypsin-Sepharose®: The pooledmaterial from the cation exchange chromatography (˜20 ml) was appliedonto an anhydrotrypsin-Sepharose column (1.6×3.6 cm) equilibrated with20 mM NaP (pH 8.0). Approximately 90% of the inhibitory active materialapplied was bound; the remainder in the flow-through was collected forrechromatography. After extensive washing of the column (˜10 columnvolumes) elution was started by addition of 100 mM KCl/HCl (pH 2.1) at aflow rate of 0.3 ml/min. Fractions were collected and neutralisedimmediately by addition of 1 M Tris. The pooled eluate was dialysedagainst 20 mM NaP (pH 8.0) over night at 4° C.

c) Chromatography on Mono S®: The dialysed eluate from the affinitychromatography was bound on a Mono S cation exchange column (0.5×5 cm)equilibrated with 20 mM NaP (pH 8.0). The column was washed with thesame buffer (˜20 ml), and eluted using a gradient from 60 to 240 mM NaClin 50 column volumes at flow rate of 1 ml/min. Fractions containinginhibitory active material were pooled (˜5 ml), aliquoted, and stored at-20° C.

2.2. Standard Analytical Methods

a) Protein Assay: Protein concentrations were determined using thebicinchoninic acid procedure [Smith Krohn, 1985] with bovine serumalbumin as standard.

b) Electrophoresis: Electrophoretic analysis of reduced and denaturedprotein was performed using 10-20% SDS-polyacrylamide gradient gels asdescribed by Laemmli [Laemmli, 1970]. Proteins were detected aftersilver staining [Heukeshoven, 1985].

c) HPLC: Samples (˜1 nmol) were loaded onto a Lichrospher RP 8 reversedphase column (120×4 mm; Merck) and eluted using a linear gradient from0% to 30% acetonitrile in 0.1% TFA at a flow rate of 1 ml/min.

d) sequence analysis:

Reduction and S-β-pyridylethylation: S-β-pyridylethylation was carriedout essentially as described by Friedman et al. [Friedman Krull, 1970].The inhibitor (1-2 nmol) was dissolved in 100 μl buffer (6 Mguanidinium-HCl, 0.25 M Tris-HCL, 1 mM EDTA, 5% (v/v) β-mercaptoethanol;pH 8.5) and incubated overnight at room temperature. After addition of 5μl 4-vinylpyridine and incubation for 90 min, the reaction was stoppedby acidification with formic acid. The S-pyridinethylated inhibitor wasdesalted by reversed phase chromatography on an Aquapore RP 300 column(2.1×30 mm; Applied Biosystems, Pfungstadt, Germany).

Oxidation of the inhibitor: A mixture of formic acid (45 μl) andhydrogen peroxide (30%; 5 μl) was preincubated for 1 h at RT.Thereafter, the inhibitor (1-2 nmol) was dissolved in this mixture.After incubation for 1 h at 4° C., the reaction was stopped by dilutionwith 1 ml deionised water and lyophylisation.

Fractionation: The inhibitor (1 nmol) was incubated with trypsin and/orchymotrypsin (both sequencing grade; Boehringer Mannheim) in 100 μl of 1M ammoniumhydrogencarbonate buffer (pH 8.0) for 14 h at 37° C. Anenzyme/inhibitor ratio of 1:40 was used. The reaction was terminated byacidification with formic acid, and fragments were separated by HPLC.

Amino acid sequence analysis: Automated amino-acid sequencing wasperformed using a gas-phase sequencer 473A (Applied Biosystems,Weiterstadt, Germany).

e) sequence comparison: The MIPSX-database (Martinsrieder Institut furProteinsequenzen am Max-Planck-Institut fur Biochemie, Martinsried,Germany) was searched using the Lipman & Pearson fast protein searchingalgorithm FASTP [Lipman Pearson, 1985]. Alignments were optimised usingCLUSTAL [Higgins Sharp, 1988].

f) Amino acid analysis: Samples of oxidised inhibitor were hydrolysedunder vacuum in 5.7 M hydrochloric acid at 110° C. for 20 h and analysedon a Biotronik LC 5000 high performance analyser system (Puchheim,Germany).

g) Determination of the molecular mass: The molecular mass of theHPLC-purified inhibitor (50 μM) was determined using a tandem quadrupoleinstrument API III (Sciex, Thornhill, Ontario, Canada). The instrumentwas calibrated with the ammonium adduct ions of polypropylene glycol.

h) Inhibitory activity: During the purification procedure the inhibitorwas followed by measurements of its effect on the amidolytic activity oftryptase. Therefore, samples were incubated with tryptase (0.59 nM) in50 mM Tris/HCl (pH 7.6), 150 mM NaCl, 50 μg/ml bovine lung heparin, and0.1% (w/v) bovine serum albumin for 25 min at 37° C. The assay wasstarted by addition of the substrate tos-Gly-Pro-Arg-pNa at a finalconcentration of 0.1 mM. The released nitroaniline was monitoredspectrometrically at 405 nm for 3.5 min using a UVIKON 930 photometer(Kontron; Eching, Germany).

One inhibition unit (IU) was defined as the amount of inhibitor whichreduces the substrate hydrolysis by 30%.

i) Titration of the inhibitor: The concentration of inhibitory activeleech-derived tryptase inhibitor was determined by titration withtrypsin. Therefore, bovine pancreatic trypsin was standardised byactive-site titration using p-Nitrophenyl p'-guanidinobenzoate [ChaseShaw, 1970]. The concentration of active inhibitor was calculatedassuming a 1:1 interaction between the inhibitor and trypsin.

k) Determination of equilibrium constants: To determine the specificityof the inhibitor, its effect on the amidolytic activity of variousserine proteinases (see Tab. 5) was determined. Therefore, proteinaseswere incubated with the inhibitor (0.2 μM) for 15 and 30 min under theconditions indicated in Tab. 5. The residual enzyme activity wasmeasured after addition of a suitable substrate.

Equilibrium dissociation constants (K_(i)) for the complexes of theinhibitor with individual proteases were determined essentially asdescribed by Bieth [Bieth, 1980]. Briefly, increasing concentrations ofthe inhibitor were incubated with a constant concentration of theenzyme; the time necessary to reach equilibration of theenzyme-inhibitor complex was determined for each protease in preliminaryexperiments. Substrate was then added, and the residual enzyme activitymeasured. K_(i) -values were calculated by fitting the steady statevelocities to the equation for tight binding inhibitors [Morrison, 1969]using non-linear regression analysis.

1) Coagulation assay: The prothrombin time according to Quick and thepartial thromboplastin time were measured using an Amelung KC 10coagulometer (Lemgo, Germany) and the reagent sets from Behringwerke AG(Marburg, Germany) according to the guidelines of the manufacturers.

m) Cleavage of vasoactive intestinal peptide (VIP): Tryptase (4.8 nM)was preincubated with different concentrations of the leech-derivedtryptase inhibitor in 100 mM Tris (pH 7.4), 140 mM NaCl, 50 μg/mlheparin at 37° C. for 25 min. Vasoactive intestinal peptide (VIP; 24 μM,final concentration) was then added. After incubation for additional 1to 10 min, the reaction was stopped by acidification with acetic acid.The remaining VIP and the fragments generated were quantified usingHPLC.

n) Growth of human keratinocytes: For growth studies the humankeratinocyte cell line HaCaT, a spontaneously transformed cell linemaintaining characteristics of differentiated keratinocytes [BoukampPetrussevska, 1988], was utilised. HaCaT cells were plated in 24-welltissue culture plates (Falcon; Becton Dickinson, Heidelberg, Germany) ata density of 10⁴ cells/cm² in a medium containing 90% Dulbecco'smodified Eagles' medium, 10% foetal calf serum, and 50 μg/ml gentamicin.Cells were incubated at 37° C. in 5% CO₂. After 24 h, the cells werewashed twice with serum-free Dulbecco's modified Eagles' medium, andfresh serum-free medium containing 7.8 μg/ml heparin alone or mediumcontaining the agonists and/or the inhibitor was added. After 48 h, thecells were washed two times again, and fresh serum-free mediumcontaining 1 μCi/ml ³ H-thymidin was added. After additional 2 h, thecells were washed three times with ice-cold Dulbecco's PBS, andincorporated ³ H-thymidin was precipitated by 10% trichloroacetic acid.After solubilisation of the precipitate in 0.1 N NaOH, 1% SDS, theincorporated radioactivity was determined by liquid scintillationcounting (beta counter model LS 1800, Beckman Instruments, Munich,Germany). For growth studies other keratinocyte cell lines may beapplied as well.

EXAMPLE 1 Isolation of the Leech-derived Tryptase-inhibitor

3.5 g of lyophilised leech extract was dissolved in water, dialysedagainst 20 mM NaP (pH 8.0), and applied onto a SP-Sephadex® cationexchange column (see Method Section). The bulk of the protein (˜98%) andof the trypsin-inhibitory activity was found in the flow-through,whereas the leech-derived tryptase inhibitor was bound to the column.After elution of the column with 500 mM NaCl (FIG. 1), the inhibitor wasseparated from non-trypsin inhibiting proteins by subsequentaffinity-chromatography on anhydrotrypsin-sepharose (FIG. 2). Finalpurification was achieved by Mono S® cation-exchange chromatography(FIG. 3). The data of the isolation procedure are summarised in Table 1.

                  TABLE 1                                                         ______________________________________                                        Purification of the leech-derived tryptase inhibitor.                                                        Specific    Purifi-                            Purification                                                                           Volume  Protein Activity                                                                            activity                                                                             Yield                                                                              cation                             step     [ml]    [mg]    [IU]  [IU/mg]                                                                              [%]  (fold)                             ______________________________________                                        Dialysis 91.5    2311    124000                                                                                50   --   --                                 SP-Sephadex                                                                            19.7    35.8    103000                                                                               2890  83    58                                Anhydro- 15.2    2.11     47100                                                                               22300 38   446                                trypsin-                                                                      Sepharose                                                                     Dialysis 14.5    1.75     36300                                                                               20700 29   414                                Mono S    4.5    0.08     9910 122000  8   2440                               ______________________________________                                    

3.5 g of lyophilised leech extract was used as starting material. Oneinhibitory unit (IU) was defined as the amount of inhibitor reducing theamidolytic activity of tryptase by 30% (see Methods).

The isolated leech-derived tryptase inhibitor was homogeneous accordingto SDS-PAGE and N-terminal sequence analysis (FIGS. 4 and 8). However,two species were separated by reversed phase HPLC (FIG. 5). Subsequentamino acid sequencing after tryptic fragmentation, amino acid analysis,and mass spectroscopy (Tab. 2, FIG. 6 and 7) demonstrated that the twospecies comprise three forms differing only in their C-terminalsequence. Thus, forms B (43 aa) and C (46 aa) differ from the shortestform A (42 aa) by a C-terminal extension of -GLY and -GLY-ILE-LEU-ASN,(the last 4 amino acids of SEQ ID NO:3) respectively. The resultsobtained for the 3 forms are compared in Tab. 3.

                  TABLE 2                                                         ______________________________________                                        Amino acid analysis of the two species of the leech-                          derived tryptase inhibitor separated by HPLC (see FIG. 5).                    Amino acid     Form A/B.sup.1)   Form C                                       ______________________________________                                        Asx            3.49   (3)        4.18 (4)                                     Thr            2.17   (2)        2.22 (2)                                     Ser            4.76   (5)        4.86 (5)                                     Glx            1.43   (1)        1.26 (1)                                     Gly            5.3    (4/5).sup.2)                                                                             5.62 (5)                                     Ala            3.43   (3)        3.18 (3)                                     Cys            5.68   (6)        5.24 (6)                                     Val            2.82   (3)        2.94 (3)                                     Ile            3.15   (3)        4.19 (4)                                     Leu            1.08   (1)        2.03 (2)                                     Tyr            n.d.   (1)        n.d. (1)                                     Lys            5.03   (5)        5.00 (5)                                     Arg            1.7    (2)        1.74 (2)                                     Pro            3.01   (3)        3.81 (3)                                     ______________________________________                                    

The values given in brackets are the values calculated from the aminoacid sequence. ¹) Forms A and B have not been separated; ²) The sequenceof the forms A and B contain 4 and 5 glycins, respectively; n.d. notdetermined

                  TABLE 3                                                         ______________________________________                                        Summary of the characterisation of the three forms of                         the leech-derived tryptase-inhibitor.                                                        Form A Form B   Form C                                         ______________________________________                                        C-terminal Sequence                                                                            CPT      CPTG     CPTGILN                                    Molecular mass determined by                                                                   4340     4396     4738                                       mass spectroscopy                                                             Molecular mass calculated                                                                      4341     4398     4738                                       from the sequence.sup.1)                                                      Elution time on reversed                                                                       25 min.sup.2) 29 min                                         phase HPLC (see FIG. 5)                                                       Elution time of the C-                                                                         13 min.sup.2) 23 min                                         terminal peptide of the                                                       tryptic digest (see FIG. 6)                                                   Inhibitory activity                                                                            +.sup.2)      +                                              ______________________________________                                         .sup.1) assuming three disulphide bonds                                       .sup.2) Forms A and B have not been separated                            

The N-terminal 35 amino acid residues of the leech derived inhibitorwere determined by sequencing the native inhibitor. The primarystructure was completed and verified using overlapping peptidesgenerated after modification and tryptic and/or chymotrypticfragmentation (FIG. 8).

Sequence comparisons demonstrate that the leech-derived tryptaseinhibitor is a non-classical Kazal-type serine proteinase inhibitor. Thehighest degree of similarity was found to Bdellin B [Fink Rehm, 1986];in the sequence section common to both inhibitors (amino acids 1-40), 19of 40 (47.5%) amino acids are identical (Tab. 4). Despite the highsequence identity to the leech-derived tryptase inhibitor, Bdellin B, aninhibitor also isolated from the medical leech, does not affect tryptase(unpublished observations).

                  TABLE 4                                                         ______________________________________                                        Comparison of the amino acid sequences of the                                 leech-derived tryptase inhibitor and Bdellin B-3                              [Fink Rehm, 1986].                                                            ______________________________________                                        Tryptase-                                                                             1   5    10   5    20   5    30   5    40                             Inhibitor                                                                             KKVCACPKILKPVCGSDGRTYANSCIARCNGVSIKSEGSC                              Bdellin DTECVCTKELHRVCGSDGVTYDNECLATCHGASVAHDHAC                              B-3     .. *.*.* *..****** **.*.*.* *.*.*.  . .*                              Tryptase-                                                                     Inhibitor                                                                     PT                                                                            Bdellin EGHEEHHVDEHGEDHD                                                      B-3                                                                           ______________________________________                                         (* = identical residues; . = homologues amino acids)                     

EXAMPLE 2 Specificity of the Leech-derived Inhibitor

The leech-derived tryptase-inhibitor inhibits human tryptase in aconcentration-dependent fashion. Using the tripeptide-nitroanilidtos-Gly-Pro-Arg-pNa as a substrate, a maximal inhibition of 50% wasobserved (FIG. 9). Thus, most likely due to steric hindrance, theinhibitor blocks only two of the four catalytic subunits of the tryptasetetramer, leaving the other two subunits accessible to the smallsubstrate. The interaction of the inhibitor with the first twotryptase-subunits can be described mathematically as a tight bindinginhibition with a K_(i) of ˜1.4 nM for the complex. The leech-derivedtryptase-inhibitor is highly specific and inhibits only trypsin andchymotrypsin with affinities similar to that for tryptase (Tab. 5). Incontrast, the K_(i) -values for the complexes with other proteinases areat least 200 times higher.

                                      TABLE 5                                     __________________________________________________________________________    Specificity of the Leech-derived tryptase inhibitor. (.sup.1) at 0.2          μM; .sup.2) K.sub.i for the                                                inhibition of two of the four subunits of the tryptase-tetramer; ni, no       inhibition at 0.2 μM;                                                      nd, not determined)                                                                                                 Enzym-                                                                            Substrate-                                                                concen-                                                                           concen-                                                                            Pre-                                                                 tration                                                                           tration                                                                            incuba-                                                                           K.sub.m                                                                          K.sub.i                                                                             K.sub.i           Enzym   Substrate         Buffer      [nM]                                                                              [mM] tion                                                                              [mM]                                                                             [nM]  [nM]              __________________________________________________________________________    Tryptase                                                                              Tos--Gly--Pro--Arg--pNA                                                                         50 mM Tris/HCl pH 7.6                                                                      0.59                                                                             0.1  25 min,                                                                           0.38                                                                             1.8   1.4.sup.2)        (human)                   150 mM NaCl, 0.1% BSA                                                                              37° C.                                            50 ug/ml BLH,                                                                 0.5% DMSO                                           Trypsin Tos--Gly--Pro--Arg--pNA                                                                         50 mM Tris/HCl pH 7.8                                                                      0.45                                                                             0.1  20 min,                                                                           0.02                                                                             4.7   0.9.sup.          (bovine)                  0.1% Triton, 1% DMSO 37° C.                  Chymotrypsin                                                                          Suc--Val--Pro--Phe--pNA                                                                         100 mM Tris/HCl pH 7.8                                                                    1.1 0.25 15 min,                                                                           0.04                                                                             137   20                (bovine)                  0.1% Triton, 1% DMSO 37° C.                  Plasmin Tos--Gly--Pro--Lys--pNA                                                                         50 mM Tris/HCl pH 7.8                                                                     2.7 0.14 30 min,                                                                           nd 30%   nd                (human)                   0.1% Triton, 1% DMSO 37° C.                                                                        inhibition.sup.1)       Tissue-Kallikrein                                                                     D--Vat--Leu--Arg--pNA                                                                           100 mM Tris/HCl pH 8.2                                                                    31  0.12 30 min,                                                                           nd 14%   nd                (porcine)                 0.1% Triton          25° C.                                                                        inhibition.sup.1)       Thrombin                                                                              Tos--Gly--Pro--Arg--pNA                                                                         100 mM Tris/HCl pH 8.2                                                                    1.8 0.14 30 min,                                                                           nd 12%   nd                (human)                   0.1% Triton, 7% DMSO 25° C.                                                                        inhibition.sup.1)       Cathepsin G                                                                           Suc--Val--Pro--Phe--pNA                                                                         100 mM Tris/HCl pH 7.5                                                                    16  0.25 30 min,                                                                           nd 11%   nd                (human)                   500 mM NaCl          25° C.                                                                        inhibition.sup.1)                                 0.1% Triton, 1% DMSO                                Plasma-Kallikrein                                                                     D--Pro--Phe--Arg--pNA                                                                           50 mM Tris/HCl pH 7.8                                                                     <<87                                                                              0.2  30 min,                                                                           nd ni.sup.1)               (human)                   2% DMSO              37° C.                  Faktor Xa                                                                             Bz--Ile--Glu--Gly--Arg--pNA                                                                     100 mM Tris/HCl pH 8.2                                                                    10  0.8  30 min,                                                                           nd ni.sup.1)               (human)                   0.1% Triton, 4% DMSO 25° C.                  Pankreatic                                                                            Suc--Ala--Ala--Ala--pNA                                                                         100 mM Tris/HCl pH 7.5                                                                    72  2    30 min,                                                                           nd ni.sup.1)               Elastase                  500 mM NaCl          25° C.                  (porcine)                 0.1% Triton, 2% DMSO                                Neutr. Elastase                                                                       MeO--Suc--Ala--Ala--Pro--Val--pNA                                                               100 mM Tris/HCl pH 7.5                                                                    4.2 1    30 min,                                                                           nd ni.sup.1)               (human)                   500 mM NaCl          25° C.                                            0.1% Triton, 2% DMSO                                Urokinase                                                                             Pyr--Gly--Arg--pNA                                                                              100 mM Tris/HCl pH 0.2                                                                    34  0.9  30 min,                                                                           nd ni.sup.1)               (human)                   0.1% Triton, 2% DMSO 25° C.                  __________________________________________________________________________

EXAMPLE 3 Biologic Characterisation

To determine whether the leech-derived tryptase inhibitor affects thecleavage of a biologically relevant substrate by tryptase, its effect onthe breakdown of vasoactive intestinal peptide (VIP) was measured. At aconcentration of 4×10⁻⁷ M, the inhibitor reduced the breakdown of VIP by66% (FIG. 10). Thus, the inhibitor blocks not only the tryptase-inducedcleavage of the peptide nitroanilid substrate tos-Gly-Pro-Arg-pNA (seeExample 2), but also that of a biologically relevant substrate.

Tryptase not only cleaves soluble proteins, but also directly interactswith cells activating cellular functions such as the growth offibroblasts and keratinocytes. To determine whether the leech-derivedtryptase inhibitor blocks these cellular effects of tryptase, its effecton the tryptase-induced growth of cultured human keratinocytes wasstudied. In the absence of the inhibitor, tryptase (10⁻⁹ M) markedlystimulated the growth of keratinocytes, increasing their ³ H-tymidinincorporation to 182±6% of the control. The leech-derived tryptaseinhibitor did not significantly affect the baseline growth suggesting alack of cytotoxic effects (Tab. 6).

                  TABLE 6                                                         ______________________________________                                        Effect of the leech-derived tryptase inhibitor on the                         proliferation of the human keratinocyte cell line HaCaT.                                     Growth rate                                                    Condition      (% of control)                                                 ______________________________________                                        Medium alone   100                                                            + Inhibitor 10.sup.-7 M                                                                       96 ± 3                                                     + Tryptase     182 ± 6                                                     + Tryptase     115 ± 6                                                     + Inhibitor 10.sup.-7 M                                                       + Tryptase     120 ± 2                                                     + Inhibitor 10.sup.-8 M                                                       ______________________________________                                    

Growth rates were calculated as the incorporation of ³ H-thymidine inthe presence of the agonist and/or inhibitor expressed as a percentageof the incorporation in medium alone. Data are given as mean±SEM, n≧2.

The inhibitor greatly reduces the tryptase-induced (10⁻⁹ M) cell growthwithout significant effect on the proliferation under baselineconditions. Thus, the inhibitor nearly completely blocks of the biologiceffect of tryptase without cytotoxic side effect.

However, the inhibitor significantly reduced the tryptase-inducedproliferation, reducing the ³ H-tymidin incorporation to 115±5% and120±2% of the control at a concentration of 10⁻⁷ M and 10⁻⁸ M,respectively. As this ³ H-tymidin incorporation is similar to thatcaused by 10⁻¹¹ M tryptase (118±4%), the data suggest that the inhibitorblocks the cellular effect of tryptase by approximately 99%.

Finally, the influence of the leech-derived tryptase inhibitor on theprothrombin time (according to Quick) and the partial thromboplastintime were measured to determine whether it interferes with the bloodcoagulation. At a concentration of 10⁻⁷ M, the inhibitor has nosignificant effect on both parameters (Tab. 7). Thus, the leech-derivedtryptase inhibitor does not significantly inhibit any of the proteasesinvolved in the blood coagulation cascade.

                  TABLE 7                                                         ______________________________________                                        Effect of the leech-derived tryptase inhibitor on the                         blood coagulation.                                                                               Partial                                                                 Quick Thromboplastin Time                                        ______________________________________                                        Control        89%     37.3 sec                                               Tryptase-Inhibitor                                                                           91%     37.1 sec                                               ______________________________________                                    

The inhibitor (100 nM) does not affect the prothrombin time according toQuick and the partial thromboplastin time, demonstrating that theenzymes involved in the coagulation cascade are not inhibited.

EXAMPLE 4 Pharmaceutical Preparation Containing the Tryptase Inhibitorfor Parenteral Administration

A solution prepared in accordance with Example 1 is dialysed against a0.9% strength NaCl solution. The concentration of the solution is thenadjusted to 1 mg/ml or 10 mg/ml by concentration or by dilution with thesame NaCl solution. These solutions are sterilised by ultrafiltration(membranes having 0.22 μm pores).

The sterilised solutions can be used for intravenous administration.

EXAMPLE 5 Preparation of Recombinant Tryptase Inhibitor

5.1. Materials

All chemicals used were obtained from Sigma, St. Louis, USA; Merck,Darmstadt, FRG; Serva, Heidelberg, FRG; Biomol, Hamburg, FRG; Roth,Karlsruhe, FRG; Braun, Melsungen, FRG; Dianova, Hamburg, FRG; Promega,Madison, USA. Restriction endonucleases and DNA-modifying enzymes werepurchased from Boehringer, Mannheim, FRG; New England Biolabs, Beverly,USA and Pharmacia-Biotech, Freiburg, FRG. Adenosine-5'-α [³⁵S]-thiotriphosphate was obtained from Amersham Buchler, Braunschweig,FRG.

Bacto-tryptone, Bacto-peptone, Bacto yeast nitrogen base (without aminoacids, w/o), Bacto yeast extract and Bacto-agar were from Difco,Augsburg, FRG. As culture media we used 2×YT [Sambrook et al, 1989]; YPD(10 g Bacto yeast extract, 20 g Bacto peptone, 20 g glucose, pH 6.0);YED (20 g Bacto yeast extract, 20 g glucose, 6.7 g NaH₂ PO₄, pH 6.0) andSD+ (6.7 g Bacto nitrogen base (w/o), 20 g glucose, 6.7 g NaH₂ PO₄, 9 mgL-leucine, pH 6.0).

Oligonucleotides were purchased from MWG-Biotech, Munchen, FRG orsynthesized by Dr. S. Modrow, Munchen,FRG.

Vectors and strains: The E. coli pUC cloning vector was from PharmaciaBiotech Europe GmbH, Freiburg. The E.coli-S.cerevisiae shuttle andexpression vector pVT102U/α and the yeast strain S-78 were kindlyprovided by T. Vernet, Montreal, CAN and by C.-W. Chi and Y.-S. Zhangboth Shanghai, China [Lit. Vernet et al, Chen et al). E. coli TG1((lac-pro), supE, thi, hsdD5/F'traD36, proA⁺ B⁺, lacI^(q), lacZM15) wasfrom Amersham-Buchler, Braunschweig,FRG,; E.coli JM105 (thi, rspL, endA,sbcB15, hspR4, (lac-proAB) F'traAB proAB, lacI^(q), lacZM15); and E.coli HB101 (F⁻, pro⁻, leu⁻, thi⁻ lacY, Sm^(r), endol⁻, recA⁻, r_(k) ⁻,m_(k) ⁻) were from Deutsche Stammsammlung Braunschweig, FRG.

The standard techniques of molecular cloning were performed according toSambrook et al. [Sambrook et al.,1989] and to M.-D. Rose et al. [Rose etal., 1990].

5.2. Standard Analytical Methods

a) SDS-PAGE and isoelectric focussing (IEF) SDS-PAGEs of the proteinswere performed with 15-25% polyacrylamide gels following the procedureof Laemmli [Laemmli, 1970]. The gels were either self-prepared and runin a conventional apparatus or in the PhastSystem (Pharmacia,Sollentuna, Sweden). Isoelectric focussing was also done with thePhastSystem using the isoelectric focussing calibration kit pH 3-10.

b) EPLC analysis, amino acid sequencing

Usually 2-3 nmol of protein were analysed by reversed phase-HPLC asdetailed previously [Auerswald et al., 1991. The N-termini weresequenced with a gas-phase sequencer 473A (Applied Biosystems GmbH,Weiterstadt, FRG) following the instructions of the manufacturer.

c) Determination of the protein concentrations

To determine the protein concentration the Pierce BCA* Protein Assaywith BSA as standard protein [Smith et al., 1985] was used. A_(280nm)(1%) was calculated for recombinant LDTI-C using the A₂₈₀ values foraromatic residues and cystines of Mach et al. [1992]: A₂₈₀ (1%)=3.46,and for protein mixtures A₂₈₀ (1%)=1.

d) Trypsin inhibition assay

The concentration of inhibitorily active material and the specificinhibitory activity of rLDTI-C was determined indirectly by measuringthe residual trypsin activity using the following conditions describedby Chase and Shaw, 1970. Test buffer: 0.05M Tris-HCl pH7.6, 150 mM NaCl.0.1% (v/v) Triton X-100, 600 pM trypsin; 100 μM Tos-Gly-Pro-Arg-p-NA.

e) Determination of K_(i) values

Equilibrium dissociation constants (K_(i)) for the complexes of rLDTI-Cwith trypsin and tryptase were determined essentially as describedpreviously [Bieth, 1980].

5.3. Construction of the Synthetic LDTI-C Gene.

A synthetic gene coding for a recombinant homologue of LDTI form C wasdesigned and constructed as outlined in FIG. 11. The DNA sequence wasselected on the basis of the amino acid sequence of natural tryptaseinhibitor by assistance of the GCG sequence analysis software [UWGCG,Devereux et al., 1984] with the E.coli and S.cerevisiae codon usages forstrongly expressed genes [Bennetzen and Hall, 1982].

The 5'--OH ends of the internal oligonucleotides were phosphorylatedusing T4 polynucleotide kinase before hybridisation. All sixoligonucleotides, 200 pmol each, were heated 5 minutes to 95° C.Hybridization was achieved during cooling down to room temperaturewithin 8 hours. After phenol extraction and ethanol precipitation,internal nicks were ligated by T4 ligase (Boehringer), according to themanufacturers protocol. The material was separated by gelelectrophoresis on a low melting agarose (5%) and a 149 bp long fragmentwas purified using the MERMAID isolation kit from Dianova, Hamburg.

5.4. Construction of Cloning Vector pRM3.1.10.

The DNA fragment obtained according to 5.3. was ligated into vectorpUC18 cut with EcoRI/HindIII (molar ratio of vector:fragment, 1:20).Competent E.coli TG1 cells were transformed with the ligation mixtureand recombinant clones were selected. DNA sequencing was performed usingthe M13/pUC (-40) sequencing primer, a 17mer, and the reversesequencings primer (-48), a 24 mer. Vector pRM3.1.10 (FIG. 15)containing the designed sequences of rLDTI-C was used for furtherexperiments.

5.5. In vitro Production and Cytoplasmic Expression in E. coli

a) The synthetic LDTI-C gene and the expression vector pASK 40 [Skerraet al., 1991] were cleaved separately with EcoRI and HindIII, purifiedand ligated. Modified pASK 40 was designated pRM4.1.4 (FIG. 16). ThepRM4.1.4 DNA was analysed by an E.coli S-30 coupled in vitrotranscription translation system, from Promega with S-35 cysteinefollowing the instructions of the manufacturers. The in vitrotranscription translation of vector pRM4.1.4 with a commerciallyavailable S-30 E.coli lysate showed two major radioactive labelledprotein bands with app. MW of 7 kDA and 5 kDa (data not shown). Theother strong band detected, seems to be β-lactamase (app. MW of 31 kDa).The 7 kDA protein band is interpretated as the uncleaved fusion proteincontaining the ompA signal sequence and LDTI-C (theoret. MW 7038 Da)whereas the 5 kDa protein (theoret. MW 5015 Da) band seems to be thecleaved and expected [ANS] LDTI-C which is prolongated by three aminoacid residues.

b) For cytoplasmic expression the synthetic LDTI-C gene was ligatedafter a fill-in reaction into pGEX-3X (Pharmacia) cleaved with SmaI. Theresulting vector was named pRM 11.1.4 (FIG. 17) and the resulting hoststrain is E.coli 1314 Cytoplasmic glutathione-S-transferase-LDTI-Cfusion proteins were found as insoluble inclusion bodies, with E.coli.1314 (HB 101 with pRM11.1.4, data not shown).

5.6. Construction of Expression Vector pRM 9.1.4

For the expression experiments with yeast the modified alpha matingsecretion system pVT102U/α ]Vernet et al., 1987) was selected in whichTrichosanthes trypsin inhibitor, a small serine proteinase inhibitor ofthe squash family was expressed successfully [Chen et al., 1992]. Withinthis system the recombinant inhibitor was correctly folded, cleaved fromthe signal sequence, protected from proteolytic degradation and it couldbe purified in two or three steps from yeast fermentation broth.

In order to use the shuttle vector pVT102U/α, the rLDTI-C gene had to bemodified first. The LDTI-C gene (FIG. 11) was mutated by substitutingthe EcoRI/SphI cassette with a XbaI/SphI linker cassette. The sequenceof this XbaI/SphI linker is CTAGATAAAAGAAAGAAGGTTTGCGCATGV (SEQ IDNO:22). It codes for the C-terminal end (FIG. 11c) of the alpha matingtype signal sequence containing a cleavage site for the KEX2 signalpeptidase (Lys Arg) and the N-terminus of LDTI. The modified LDTI-C genewas assembled via a three fragment ligation using the XbaI/SphI linkercassette, the SphI/HindIII LDTI-C fragment and the XbaI/HindIII cleavedpUC18 vector (molar ratio 10:5:1). After transformation of E.coli TG1recombinant clones were screened by restriction analysis and DNAsequencing using the M13/pUC (-40) primer (Biolabs) and the M13/pUC/-48)reverse primer (Biolabs) (CGCAGTAGCGGTAAACG) (SEQ ID NO:24. The newvector pRM 5.1.5 (FIG. 12a) carried the expected sequence and theXbaI/HindIII fragment including the rLDTI-C gene was ligated intoXbaI/HindIII cleaved pVT102U/α. The resulting expression vector pRM9.1.4 (FIG. 12b) was isolated and used to transform S.cerevisiae strainS-78 according to the method of Becker and Guarante [Becker andGuartante, 1991].

5.7. Expression in Saccharomyces cerevisiae

Analytical rLDTI-C expression experiments using yeast strain H005 (S-78with pRM9.1.4) were carried out with Fernbach flasks (180-220 rpm, 28°C.; pre-culture for 3 days with 100 ml SD(+) media and main cultures for4 days with 900 ml fresh YED-media). At each day cell density (OD₇₀₀)was determined, pH was adjusted to 6.0 with 1M NaOH, 10 ml yeast extractstock solution 50% and 30 ml 50% (w/v) glucose were added and theinhibition of trypsin was determined.

After transformation of competent S-78 strains with pRM9.1.4 expressionof rLDTI was detected. The broth of cultivated recombinant yeast cellsshowed remarkable trypsin inhibition The concentrated supernatant gave aprotein pattern with the strongest band migrating at an app. MW of 5000Da after SDS-PAGE (see FIG. 13, lane 2).

The recombinant material was isolated preparatively from culture brothof S. cervisiae cultivated in 1 liter shaker-flasks for 96 h. After thistime the growth curve of yeast cells reached an OD₇₀₀ of 22.0. Trypsininhibitory activity was detected after two days and increased parallelto the biomass.

The yeast broth was harvested (6000 g, 20 min, 4° C.) after 96 hcultivation and the supernatant was filtered, first through a 0,16 μmmembrane and then through a crossflow membrane with a 3 kDa cut-off(Filtron Omega Minisette, Filtron, Karlstein, FRG)). The buffer wasexchanged by diafiltration to 20 mM NaH₂ PO₄ pH 8.2. The material waspurified by cation-exchange chromatography (Fractogel EMD SO₃ ⁻ 65o (S)column 150-10; Merck), flow rate 3 ml/min, elution buffer 20 mM NaH₂PO₄, pH8.2, 500 mM NaCl.

The data of a representative purification are summarized in Table 8.

                  TABLE 8                                                         ______________________________________                                        Results of a typical purification of r LDTI form C                            from Saccharomyces cereviseae culture supernatant                                                          Active Specific                                             Volume  Total protein                                                                           material                                                                             activity                                                                            Yield                               Purification step                                                                        (ml)    (mg)      (mg)   (%)   (%)                                 ______________________________________                                        culture supernatant                                                                      1000    5620      10.6   0.1   100                                 culture supernatant,                                                                     965     4600      5.2    0.1   49                                  0.16 μm filtration                                                         retentate, 3K-                                                                           125      850      4.6    0.5   43                                  membrane                                                                      Fractogel EMD SO.sub.3                                                                    21       5       3.2    59    30                                  main fractions                                                                ______________________________________                                    

Total protein was estimated applying the Pierce assay (bovine serumalbumin as standard); active material was calculated from trypsininhibitory assays; yield is given as percentage of isolated material;

From one liter fermentation broth 3 mg rLDTI-C were obtained. TheSDS-PAGE of this material showed a homogeneous but relatively broad bandmigrating at an app. MW of 5000 Da (FIG. 13, lane 3). About 85% ofrLDTI-C eluted as a sharp peak at 28% acetonitrile when analysed by RP18HPLC. The amino acid sequencing of peak 1 (FIG. 14) revealed theexpected N-terminus KKVCACPK. But small heterogeneities were observedafter RP-HPLC and a different N-terminus was identified (peak 2)starting with 11 additional amino acids of C-terminal part of alphafactor signal peptide (FIG. 14).

This demonstrates that the endogeneous KEX 2 protease of yeast didcleave with high accuracy after LysArg, the recognition site of thesignal peptidase KEX2, and still in front of the two N-terminal aminoacid residues LysLys of LDTI. Isoelectric focussing with the PhastSystemdemonstrated that the isoelectric point of rLDTI was above pH 10.

The determined inhibition constants of the complexes tryptase-LDTI-C andtrypsin-LDTI-C are similar to those with natural LDTI. The measuredspecific trypsin inhibitory activity of 60% is comparable to otherrecombinant inhibitors.

rLDTI-C inhibits human tryptase in a fashion similar to the naturallyoccuring leech-derived tryptase inhibitor: using thetripeptide-nitroanilid tos-Gly-Pro-Arg-pNa as a substrate, a maximalinhibition of ˜50% was observed, and a K_(i) of 1.9 nM was calculatedfor the complex between tryptase and rLDTI-C.

EXAMPLE 6 Construction of pFBY166

pFBY166 is a pUC18 derived plasmid that contains a 1085 bp BamHIfragment. This fragment contains the CUP1 promoter fused to the ATG ofthe α-factor leader, a stuffer fragment and the α-factor terminator. Theprecise way the fusions were engineered enable the insertion of ORF(open reading frame) containing fragments either at the ATG by using theEcoRI site, after the signal sequence by using a PstI site or after theα-factor leader sequence by insertion after the BglII site. The ORF tobe expressed should ideally have a SalI site at their 3' end tofacilitate fusion to the terminator that is preceded by a SalI site, andhave no BamHI sites within their sequence, as cleavage of this plasmidat the two BamHI sites excises the whole expression cassette so that itcan easily be cloned into a yeast shuttle vector.

pFBY166 contains a 425 bp BamHI/EcoRI fragment of the CUP1 promoter,corresponding to nucleotides 1080 to 1505 of EMBL GENBANK accessionnumber K02204. The CUP1 promoter allows expression in a copper regulatedmanner.

The ATG is provided as part of the α-1 factor pheromone signal sequenceand leader, nucleotides 293 to 527 of the EMBL GENBANK accession numberX01581 followed by the sequence, AGATCTTGC, which positions a BglIIsite, which is unique in pFBY139, just before the normal position forthe LysArg KEX2 cleavage site. If fusions are required to just a signalsequence this can be achieved by using the unique PstI site which ispresent within the region encoding the signal sequence. The BglII siteis followed by a sequence of no importance as it is always removed whenthe incoming ORF is cloned into the plasmid between either the EcoRI,PstI or BglII sites and the SalI site which marks the end of the stufferfragment and the beginning of the α-1 factor pheromone terminatorsequences, nucleotides 825 to 1100 of EMBL GENBANK accession numberX01581. This is followed immediately by the sequence AATTCGGATCC (SEQ IDNO:23) which encodes the BamHI site that bounds this end of theexpression cassette.

This plasmid can be constructed using polymerase chain reaction (PCR)fragments from yeast genomic DNA.

All oligonucleotides used in the PCR reaction are synthesized using anautomatic DNA synthesizer. The PCR reactions are carried out in a PCRunit from Perkin Elmer under the following conditions: 20 mM of theoligonucleotides in question are incubated in 0.1 ml buffer (10 mMTris-HCl, pH 8.3, 50 mM KCl , 1.5 mM MgCl2) with 2.5 units of TaqDNA-polymerase and 0.2 mM of dATP, dCTP, dTTP and dGTP. The reactionsare incubated for 30 cycles: 30 sec at 92° C., for 1 min at 42° C. andat 72° C. for 1 min.

The fragment comprising most of the α-factor signal and leader sequencesis generated from genomic yeast DNA using the PCR fragments 1 (SEQ IDNO: 10) and 2 (SEQ ID NO: 11):

                                1. 5' GTGCGAATTCAAAATGAGATTTCCTTCAATTTTTACTGCA                                G 3'                                                                          2. 5' CAAAGTCGACTTTATCCAGCAAGATCTCTTCTTCTTTAGC                                AGCAATGC 3'                                   

The fragment comprising the α-factor terminator is generated fromgenomic yeast DNA using the PCR fragments 3 (SEQ ID NO: 12) and 4 (SEQID NO: 13):

    3. 5' GAAGAGATCTTGCTGGATAAAGTCGACTTTGTTCCCACTGTACTTTTAGC 3'                   4. 5' CCGGGGATCCGAATTAATTCTCTTAGGATTCG 3'                                 

The fragment comprising the CUP1 promoter is generated from genomicyeast DNA using the PCR fragments 5 (SEQ ID NO: 14) and 6 (SEQ ID NO:15):

    5. 5' TAGAGGATCCCCATTACCGACATTTGGGCGCTATACGTGC 3'                             6. 5' CGACGAATTCACAGTTTGTTTTTCTTAATATCTATTTCG 3'                          

and subsequent cleavage with BamHI and EcoRI.

The fragment comprising most of the α-factor signal and leader sequencesand the fragment comprising the α-factor terminator are mixed andreamplified in a PCR reaction with oligonucleotide 1 and oligonucleotide3 and cut with EcoRI and BamHI. The later amplified fragment and thefragment comprising the CUP1 promoter are cloned into pTZ18R cut withBamHI and treated with bacterial alkaline phosphatase to create pFBY139.

EXAMPLE 7 Construction of pHE 174 Expression of Tryptase Inhibitor UnderControl of the Regulated CUP1 Promoter

A synthetic gene encoding tryptase inhibitor in preferred yeast codonusage is assembled from 3 synthetic oligonucleotides in a PCR reaction.In addition, the gene is extended at its 5' end to provide forconvenient in-frame fusion to the α-factor leader in plasmid pFBY 166.

The following 3 oligonucleotides are synthesized using an automatic DNAsynthesizer:

    (SEQ ID NO: 16)                                                               1. 5'-AAAGATCTTG CTGGATAAAA GAAAGAAGGT TTGCGCCTGT                                CCAAAGATTT TGAAGCCAGT TTGTGGTTCT GACGGTCGTA                                   CC-3'                                                                      (SEQ ID NO: 17)                                                               2. 5'-ACAAGAACT TCAGACTTAA TAGAAACACC GTTACAACGG                                 GCAATACAAG AGTTGGCGTA GGTACGACCG TCAGAACCAC-3'                             (SEQ ID NO: 18)                                                               3. 5'-TTGTCGACTC AGTTCAAAAT ACCGGTTGGA CAAGAACCTT                                CAGACTTAA-3'                                                           

Of these 3 oligonucleotides a 170 bp fragment is assembled in thefollowing polymerase chain reaction (PCR) using the PCR unit from PerkinElmer and the following conditions:

20 mM of oligonucleotides 1 and 3 and 20 nM of oligonucleotide 2 areincubated in 0.1 ml buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mMMgCl2) with 2.5 units of Taq DNA-polymerase and 0.2 mM of dATP, dCTP,dTTP and dGTP. The reaction is incubated for 30 cycles: 30 sec at 92°C., for 1 min at 42° C. and at 72° C. for 1 min.

The 170 bp PCR fragment is isolated over a 2% agarose gel, restrictedwith BglII and SalI and ligated into BglII and SalI cut pFBY 166(supra). E. coli HB101 is transformed with the resulting plasmid pHE174.The transformed E. coli strain is designated E. coli/pHE174.

Correct fusion of the PCR fragment to the α-factor leader and correctsequence of the tryptase inhibitor ORF is confirmed by sequencing.

EXAMPLE 8 Construction of pHE 175 and 175R 2 Micron Vectors with theTryptase Inhibitor Expression Cassette

For the expression in yeast pDP34 is used as vector. pDP34 (EP-A-340170, FIG. 3 therein) is a yeast-E. coli shuttle vector with theampicillin resistance marker for E. coli and the URA3 and dLEU2 yeastselective markers. It contains the complete 2 micron sequences in the Aform and is REP1, REP2 and FLP proficient.

Plasmid pDP34 is digested with BamHI and the sticky ends aredephosphorylated by alkaline phosphatase treatment. pHE174 is digestedwith BamHI and the 1119 bp fragment containing the complete tryptaseinhibitor expression cassette ligated into BamHI-cut pDP 34. E. coli HB101 is transformed with the resulting plasmids pHE 175 and 175R.Orientation of the insert is tested by digestion with SalI. pHE 175contains the Tryptase inhibitor expression cassette in a clockwiseorientation with respect to dLEU2, pHE 175R in anticlockwise orientationwith respect to the dLEU2 marker.

EXAMPLE 9 Construction of pHE 176 The Tryptase Inhibitor ORF Fused tothe Invertase Signal Sequence (SUC2)

To provide for an alternative secretion system, the tryptase inhibitorORF is fused to the signal sequence of the yeast invertase gene SUC2.

The 2 following oligonucleotides are made:

    (SEQ ID NO: 19)                                                               1. 5'-TTGTCGACTC AGTTCAAAAT A-3'                                              (SEQ ID NO: 20)                                                               2. 5'-AAGAATTCAT GCTTTTGCAA GCTTTCCTTT TCCTTTTGGC                                TGGTTTTGCA GCCAAAATAT CTGCAAAGAA GGTTTGCGCC                                   TGTC-3'                                                                

pHE 174 is used as template DNA for a polymerase chain reaction asdescribed in example 7. 20 ng of template pHE 174 is incubated with 20mM of the oligonucleotide primers under the experimental conditions asin example 7.

The 214 bp amplified PCR fragment is isolated over a 2% agarose gel,restricted with EcoRI and SalI and ligated into EcoRI and SalI cutvector pFBY 166.

E. coli HB 101 is transformed with the resulting plasmid pHE 176.Correct sequence of the SUC2 signal sequence-tryptase inhibitor fusionis confirmed by sequencing.

EXAMPLE 10 Construction of pHE 177 and pHE 177R 2 Micron Vectors withthe Tryptase Inhibitor Expression Cassette with the SUC2 Signal Sequence

In analogy to example 8, the 918 bp BamHI fragment containing thetryptase inhibitor expression cassette is excised from pHE 176 by BamHIdigestion and inserted into BamHI cut pDP 34. E. coli HB 101 istransformed with the resulting plasmids pHE 177 and pHE 177R.Orientation of the insert is tested by digestion with SalI. pHE 177contains the tryptase inhibitor expression cassette in a clockwiseorientation with respect to dLEU2, pHE 177R in an anticlockwiseorientation.

EXAMPLE 11 Construction of Saccharomyces cerevisiae Strain TR 1456

Saccharomyces cerevisiae strain TR1456 is constructed as disclosed inEP-A-341 215. Starting with Saccharomyces cerevisiae strain H449 (DSM4413, MATa, leu23,112, ura3, prb1 [cir.sup.° ]), in two subsequentseries of experiments the two carboxypeptidases yscα and yscY areremoved from strain H449 by disruption of their encoding genes KEX1 andPRC1, respectively. First, the gene encoding ysca, KEX1, is disrupted.

For this purpose, strain H449 is transformed with a DNA fragmentencoding the KEX1 gene, with the full URA3 gene inserted in the middleof the KEX1 coding region. Uracil prototrophic transformants areselected and tested for the absence of yscα activity. Next, the URA3gene inserted at the KEX1 locus is disrupted by transformation with aplasmid containing a disrupted version of the gene, URA3Δ5 (see EP-A-341215). Transformants which are uracil auxotrophic are selected and in thefollowing step disrupted in their endogenous PRC1 gene coding for thecarboxypeptidase yscY. The experiment is carried out in a totallyanalogous manner as described for the disruption of KEX1. The finallyresulting isogenic derivative of strain H449 is called TR1456 and hasthe following genotype:

TR1456=MATa, leu2-3, 112, ura3, prb1, kex1::ura3, prc1::ura3, [cir.sup.°]

EXAMPLE 12 Transformation of Strain TR 1456 with Plasmids pHE 175, 175R,177 and 177R

The plasmids pHE 175, 175R, 177 and 177R are introduced into the hoststrains H449 and TR1456, resp., using the transformation protocoldescribed by Hinnen et al. (Proc. Natl. Acad. Sci. USA (1978), 75,1929). Further details of the procedure are as described in EP-A-341215. Transformed yeast cells are selected on yeast minimal medium,supplemented with leucine and lacking uracil. Single transformed yeastclones are isolated and referred to as:

Saccharomyces cerevisiae TR 1456/pHE 175

Saccharomyces cerevisiae TR 1456/pHE 175R

Saccharomyces cerevisiae TR 1456/pHE 177

Saccharomyces cerevisiae TR 1456/pHE 177R

Saccharomyces cerevisiae H449/pHE 175

Saccharomyces cerevisiae H449/pHE 175R

Saccharomyces cerevisiae H449/pHE 177

Saccharomyces cerevisiae H449 /pHE 177R

EXAMPLE 13 Secretion of Leech-derived Tryptase Inhibitor by TR 1456Transformed with Plasmid pHE 177

Cells of Saccharomyces cerevisiae TR 1456/pHE 177 are grown in twosubsequent precultures of 20 ml each. The synthetic medium is composedof:

    ______________________________________                                        6.7     g/l      Difco Yeast Nitrogen Base (without                                            amino acids)                                                 10      g/l      L-asparagine                                                 1       g/l      L-histidine                                                  20      g/l      glucose                                                      0.02    g/l      L-leucine                                                    ______________________________________                                    

The pH of the medium is adjusted to 5.8. The first preculture is grownfor 60 h at 28° C. and 180 r.p.m. The second preculture is inoculatedwith 2% (v/v) of the first preculture and incubated for 24 h at 28° C.and 180 r.p.m.

The medium of the main culture is composed of:

    ______________________________________                                        5        g/l       peptone                                                    10       g/l       yeast extract                                              20       g/l       glucose                                                    40       g/l       sucrose                                                    3        g/l       ammonium sulfate                                           2        q/1       potassium dihydrogenphosphate                              0.5      g/l       magnesium sulfate heptahydrate                             0.1      g/l       sodium chloride                                            0.1      q/1       calcium chloride                                           10.sup.-5                                                                              g/l       biotin                                                     ______________________________________                                    

The main culture (100 ml medium) is inoculated with about 106 cells/mland incubated for 72 h at 28° C. and 180 r.p.m.

Immediately following the inoculation, sterile copper sulfate is addedat a concentration of 1 mM to the culture.

At the end of the fermentation, aliquots of the culture are taken, thecells are removed by centrifugation and the culture supernatant isanalyzed for activity of the leech-derived tryptase inhibitor bytitration of the inhibitor with trypsin as described under 2.2. i).

EXAMPLE 14 Analytics of the Leech-derived Tryptase Inhibitor fromFermentation Cultures of Saccharomyces cerevisiae Strain TR 1456/pRE177Using Reversed Phase HPLC

Samples from culture supernatants of strain TR 1456/pHE177 are subjectedto HPLC analysis under the following conditions:

A Merck Lichrospher 1000 RP-8 column (4×250 mm, 10 um) is used. Mobilephase A is made from water (Nanopur®, Barnstead) containing 0.1% (v/v)trifluoroacetic acid. Mobile phase B is made from 20% water (Nanopur®,Barnstead) and 80% of acetonitrile (HPLC-grade, Fluka) containing 0.09%(v/v) of trifluoroacetic acid.

Chromatographic separations are performed at a flow rate of 1.5 ml/minrunning the following gradient (Table 9). The eluents are monitored byabsorbance at 214 nm.

                  TABLE 9                                                         ______________________________________                                        t (min)          % A    % B                                                   ______________________________________                                         0               85     15                                                     8               85     15                                                    28               65     35                                                    34                0     100                                                   37                0     100                                                   42               85     15                                                    46               85     15                                                    ______________________________________                                    

One major peak with a retention time of 19.05 min is observed on thechromatogram that is present in strains bearing the inhibitor expressionplasmid, but absent in untransformed strains. Further analysis revealedthat this peak contains one species of the leech-derived tryptaseinhibitor with an apparent Mr of 4738,8 as detected by massspectroscopy. This value is in good agreement with the calculated Mrvalue of 4738, pointing to the full-length inhibitor molecule carryingboth, the correct N-terminus and C-terminus.

The chemical molecular weight of the inhibitor is determined by matrixassisted laser desorption ionization mass spectrometry (MALDI-MS) usinga home-built instrument (Boernsen et al., Chimica (1990) 44, 412-416).

In the context of the present invention the following microorganismshave been deposited with DSM (Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Braunschweig, Germany) on Jun. 29, 1994:

    ______________________________________                                        microorganism accession number                                                ______________________________________                                        pRM  3.1.10   DSM 9268                                                        pRM  4.1.4    DSM 9269                                                        pRM  5.1.5    DSM 9270                                                        pRM  9.1.4    DSM 9271                                                        pRM 11.1.4    DSM 9272                                                        HOO5          DSM 9273                                                        ______________________________________                                    

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13. Caughey, G. H., Lazarus, S. C., Viro, N. F., Gold, W. M. & Nadel, J.A. (1988) Immunology 63, 339-44.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 24                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 42 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   -      Lys Lys Val Cys Ala Cys Pro Lys - # Ile Leu Lys Pro Val Cys Gly        Ser                                                                           #   15                                                                        -      Asp Gly Arg Thr Tyr Ala Asn Ser - # Cys Ile Ala Arg Cys Asn Gly        Val                                                                           #                 30                                                          -      Ser Ile Lys Ser Glu Gly Ser Cys - # Pro Thr                            #             40                                                              - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 43 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   -      Lys Lys Val Cys Ala Cys Pro Lys - # Ile Leu Lys Pro Val Cys Gly        Ser                                                                           #   15                                                                        -      Asp Gly Arg Thr Tyr Ala Asn Ser - # Cys Ile Ala Arg Cys Asn Gly        Val                                                                           #                 30                                                          -      Ser Ile Lys Ser Glu Gly Ser Cys - # Pro Thr Gly                        #             40                                                              - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 46 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: NO                                                   #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   -      Lys Lys Val Cys Ala Cys Pro Lys - # Ile Leu Lys Pro Val Cys Gly        Ser                                                                           #   15                                                                        -      Asp Gly Arg Thr Tyr Ala Asn Ser - # Cys Ile Ala Arg Cys Asn Gly        Val                                                                           #                 30                                                          -      Ser Ile Lys Ser Glu Gly Ser Cys - # Pro Thr Gly Ile Leu Asn            #             45                                                              - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 126 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - AARAARGTNT GYGCNTGYCC NAARATHYTN AARCCNGTNT GYGGNWSNGA YG - #GNMGNACN         60                                                                          - TAYGCNAAYW SNTGYATHGC NMGNTGYAAY GGNGTNWSNA THAARWSNGA RG - #GNWSNTGY        120                                                                          #          126                                                                - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 129 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - AARAARGTNT GYGCNTGYCC NAARATHYTN AARCCNGTNT GYGGNWSNGA YG - #GNMGNACN         60                                                                          - TAYGCNAAYW SNTGYATHGC NMGNTGYAAY GGNGTNWSNA THAARWSNGA RG - #GNWSNTGY        120                                                                          #        129                                                                  - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 138 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - AARAARGTNT GYGCNTGYCC NAARATHYTN AARCCNGTNT GYGGNWSNGA YG - #GNMGNACN         60                                                                          - TAYGCNAAYW SNTGYATHGC NMGNTGYAAY GGNGTNWSNA THAARWSNGA RG - #GNWSNTGY        120                                                                          # 138              AY                                                         - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 149 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ATTTCGAAGA AGGTTTGCGC ATGCCCAAAG ATCTTGAAGC CAGTCTGTGG TT - #CTGACGGT         60                                                                          - CGTACATATG CTAACTCATG CATCGCTCGT TGTAACGGTG TATCGATCAA GT - #CTGAAGGT        120                                                                          #           149    TTTT AAACTAATA                                             - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 149 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: YES                                                    -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - AGCTTATTAG TTTAAGTTAC CGGTTGGACA AGAACCTTCA GACTTGATCG AT - #ACACCGTT         60                                                                          - ACAACGAGCG ATGCATGAGT TAGCATATGT ACGACCGTCA GAACCACAGA CT - #GGCTTCAA        120                                                                          #           149    CAAA CCTTCTTCG                                             - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 929 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 435..440                                              #/function= "EcoRI site"RMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 642..647                                              #/function= "SalI site"ORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: promoter                                                        (B) LOCATION: 1..443                                                #/phenotype= "CUP1 promoter"ION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 444..500                                              #/function= "SUC2 invertase signal                                                           sequence"                                                      -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 501..641                                              #/product= "tryptase inhibitor":                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: terminator                                                      (B) LOCATION: 648..923                                              #/standard.sub.-- name= "alpha-factor                                                        terminator"                                                    -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 924..929                                              #/function= "BamHI site"RMATION:                                              #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - GATCCCCATT ACCGACATTT GGGCGCTATA CGTGCATATG TTCATGTATG TA - #TCTGTATT         60                                                                          - TAAAACACTT TTGTATTATT TTTCCTCATA TATGTGTATA GGTTTATACG GA - #TGATTTAA        120                                                                          - TTATTACTTC ACCACCCTTT ATTTCAGGCT GATATCTTAG CCTTGTTACT AG - #TTAGAAAA        180                                                                          - AGACATTTTT GCTGTCAGTC ACTGTCAAGA GATTCTTTTG CTGGCATTTC TT - #CTAGAAGC        240                                                                          - AAAAAGAGCG ATGCGTCTTT TCCGCTGAAC CGTTCCAGCA AAAAAGACTA CC - #AACGCAAT        300                                                                          - ATGGATTGTC AGAATCATAT AAAAGAGAAG CAAATAACTC CTTGTCTTGT AT - #CAATTGCA        360                                                                          - TTATAATATC TTCTTGTTAG TGCAATATCA TATAGAAGTC ATCGAAATAG AT - #ATTAAGAA        420                                                                          - AAACAAACTG TAACGAATTC AAAATGCTTT TGCAAGCTTT CCTTTTCCTT TT - #GGCTGGTT        480                                                                          - TTGCAGCCAA AATATCTGCA AAGAAGGTTT GCGCCTGTCC AAAGATTTTG AA - #GCCAGTTT        540                                                                          - GTGGTTCTGA CGGTCGTACC TACGCCAACT CTTGTATTGC CCGTTGTAAC GG - #TGTTTCTA        600                                                                          - TTAAGTCTGA AGGTTCTTGT CCAACCGGTA TTTTGAACTG AGTCGACTTT GT - #TCCCACTG        660                                                                          - TACTTTTAGC TCGTACAAAA TACAATATAC TTTTCATTTC TCCGTAAACA AC - #ATGTTTTC        720                                                                          - CCATGTAATA TCCTTTTCTA TTTTTCGTTC CGTTACCAAC TTTACACATA CT - #TTATATAG        780                                                                          - CTATTCACTT CTATACACTA AAAAACTAAG ACAATTTTAA TTTTGCTGCC TG - #CCATATTT        840                                                                          - CAATTTGTTA TAAATTCCTA TAATTTATCC TATTAGTAGC TAAAAAAAGA TG - #AATGTGAA        900                                                                          #           929    TTAA TTCGGATCC                                             - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #   41             AGAT TTCCTTCAAT TTTTACTGCA G                               - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #                48CAGC AAGATCTCTT CTTCTTTAGC AGCAATGC                        - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #              50GGATAA AGTCGACTTT GTTCCCACTG TACTTTTAGC                      - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #          32      ATTCT CTTAGGATT CG                                         - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #    40            CCGA CATTTGGGCG CTATACGTGC                                 - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #    39            TGTT TTTCTTAATA TCTATTTCG                                  - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 82 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - AAAGATCTTG CTGGATAAAA GAAAGAAGGT TTGCGCCTGT CCAAAGATTT TG - #AAGCCAGT         60                                                                          #                 82GTA CC                                                    - (2) INFORMATION FOR SEQ ID NO: 17:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 79 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - ACAAGAACTT CAGACTTAAT AGAAACACCG TTACAACGGG CAATACAAGA GT - #TGGCGTAG         60                                                                          # 79               CAC                                                        - (2) INFORMATION FOR SEQ ID NO: 18:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 49 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #               49AAAAT ACCGGTTGGA CAAGAACCTT CAGACTTAA                       - (2) INFORMATION FOR SEQ ID NO: 19:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                AAAT A                                                     - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 84 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - AAGAATTCAT GCTTTTGCAA GCTTTCCTTT TCCTTTTGGC TGGTTTTGCA GC - #CAAAATAT         60                                                                          #                84CGCC TGTC                                                  - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: YES                                                  #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   -      Cys Pro Lys Ile Leu Lys Pro Val - # Xaa Gly Ser Asp Gly Arg Thr        #   15                                                                        -      Tyr Ala Asn Ser Cys Ile Ala                                            #   20                                                                        - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #           30     AGGT TTGCGCATGV                                            - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 11 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #       11                                                                    - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -    (iii) HYPOTHETICAL: YES                                                  -    (iii) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Hirudo me - #dicinalis                                #24:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #   17             G                                                          __________________________________________________________________________

We claim:
 1. An isolated and purified polypeptide tryptase inhibitorcomprising the amino acidsequence:Lys-Lys-Val-Cys-Ala-Cys-Pro-Lys-lle-Leu 10Lys-Pro-Val-Cys-Gly-Ser-Asp-Gly-Arg-Thr 20Tyr-Ala-Asn-Ser-Cys-lle-Ala-Arg-Cys-Asn 30Gly-Val-Ser-lle-Lys-Ser-Glu-Gly-Ser-Cys 40 Pro-Thr-X 42wherein thec-terminal residue X represents H (SEQ ID NO:1) -Gly (SEQ ID NO:2) or-Gly-lle-Leu-Asn (SEQ ID NO:3).
 2. An isolated and purified polypeptidetryptase inhibitor comprising the amino acid sequence SEQ ID NO: 21R¹-Cys-Pro-Lys-lle-Leu Lys-Pro-Val-Z-Gly-Ser-Asp-Gly-Arg-ThrTyr-Ala-Asn-Ser-Cys-lle-Ala-R² whereinthe N-terminal residue R¹represents Ala- or Cys-Ala-; the C-terminal residue R² represents -Argor -Arg-Cys; and Z defines any amino acid.
 3. A pharmaceuticalcomposition comprising a tryptase inhibiting amount of a polypeptideaccording to claims 1 and 2 optionally in combination with apharmaceutically acceptable carrier of diluent.
 4. The expressioncassette comprising a promoter operably linked to a first DNA sequenceencoding a signal peptide linked in the proper reading frame to a secondpolypeptide encoding DNA sequence of SEQ ID NO:9 and a DNA sequencecontaining transcription termination signals.
 5. The expression cassetteaccording to claim 4 wherein the promoter is selected from the groupconsisting of CUP1 p, and GAPDHp.
 6. The expression cassette accordingto claim 4 wherein the signal sequence is selected from the groupconsisting of the α-factor leader, PHO5, and SUC2.
 7. The expressioncassette according to claim 4 wherein the terminator is selected fromthe group consisting of the α-factor terminator and PHO5 terminator. 8.A vector for the transformation of eukaryotic or prokaryotic hostscomprising an expression cassette as defined in claim
 4. 9. The vectorof claim 8 which is a two-micron based yeast vector.
 10. The vector ofclaim 8 selected froma) pRM9.1.4 as deposited with the DSM and havingthe accession number DSM 9271; b) pRM11.1.4 as deposited with the DSMand having the accession number DSM 9272; c) pRM5.1.5 as deposited withthe DSM and having the accession number DSM 9270; d) pRM4.1.4 asdeposited with the DSM and having the accession number DSM 9269; and e)pRM3.1.10 as deposited with the DSM and having the accession number DSM9268.